This study investigates the composition and metabolism of microbial communities in soil pores, revealing how long-term vegetation history influences spatial distribution patterns of soil pore and particulate organic matter (POM) drivers of microbial habitats. Using single- and triple-energy X-ray computed microtomography (μCT) combined with stable isotope probing (SIP), the researchers demonstrate differences in microbial diversity, composition, and life strategies between large (30-150 μm) and small (4-10 μm) soil pores. They propose a microbial habitat classification based on biogeochemical mechanisms and soil process localization, suggesting interventions to mitigate environmental consequences of agricultural management. Soil is a critical component of terrestrial ecosystems, a major source of food production, and a key mediator of atmospheric CO₂ levels. Soil functioning is enabled by a complex microbial community inhabiting an intricate soil pore structure, adapting to variable micro-environmental conditions. Plant roots significantly influence soil microenvironments, with vegetation diversity affecting microhabitat formation. Root systems modify pore structure, while pore structure impacts soil microorganisms. Greater plant species richness increases root biomass and diversifies root residue inputs, providing more organic C for soil microorganisms and increasing microbial activity. Pore structure and water fluxes define oxygen and nutrient supply for microbial functioning. Pores <10 μm are often water-saturated, limiting oxygen supply, while pores >1000 μm are mostly unsaturated. Pores in the few-tens to couple-hundred-micron size range provide an optimal balance of oxygen, water, carbon, and nutrients for resident microorganisms. Microbial communities in different pore sizes differ in composition, life strategies, and activities. Larger pores can better stimulate fast decomposition of newly added C and have a greater abundance of certain microbial taxa compared to smaller pores. The study documents the combined effects of long-term vegetation history on soil pore and POM spatial distribution and hydraulic connectivity in small and large pores. It tests how vegetation history affects bacterial richness, community composition, and metabolism in small and large pores, with consequent effects on soil C processing. Three vegetation systems were tested: a multiyear fallow followed by monoculture corn, a perennial switchgrass community, and a polyculture restored prairie community. μCT was used to characterize soil pore structure and localize POM, with triple-energy μCT examining pore-level water distribution patterns. Labile substrate additions were simulated using labeled glucose, and SIP was used to identify microorganisms assimilating glucose-derived C. Results show that microbial habitats defined by soil pores and POM differ in ways that strongly influence microbial composition and activity, impacting ecosystem processes like decomposition, nitrogen processing, and carbon sequestration. The study highlights the importance of pore-scale hydraulic connectivity in influencing bacterial access to organic matter and subsequent metabolism, with implications for soil carbon accretion and stability. The findings suggest that microbial habitats differ in ecological C-acquisition strategies, with distinct functional rolesThis study investigates the composition and metabolism of microbial communities in soil pores, revealing how long-term vegetation history influences spatial distribution patterns of soil pore and particulate organic matter (POM) drivers of microbial habitats. Using single- and triple-energy X-ray computed microtomography (μCT) combined with stable isotope probing (SIP), the researchers demonstrate differences in microbial diversity, composition, and life strategies between large (30-150 μm) and small (4-10 μm) soil pores. They propose a microbial habitat classification based on biogeochemical mechanisms and soil process localization, suggesting interventions to mitigate environmental consequences of agricultural management. Soil is a critical component of terrestrial ecosystems, a major source of food production, and a key mediator of atmospheric CO₂ levels. Soil functioning is enabled by a complex microbial community inhabiting an intricate soil pore structure, adapting to variable micro-environmental conditions. Plant roots significantly influence soil microenvironments, with vegetation diversity affecting microhabitat formation. Root systems modify pore structure, while pore structure impacts soil microorganisms. Greater plant species richness increases root biomass and diversifies root residue inputs, providing more organic C for soil microorganisms and increasing microbial activity. Pore structure and water fluxes define oxygen and nutrient supply for microbial functioning. Pores <10 μm are often water-saturated, limiting oxygen supply, while pores >1000 μm are mostly unsaturated. Pores in the few-tens to couple-hundred-micron size range provide an optimal balance of oxygen, water, carbon, and nutrients for resident microorganisms. Microbial communities in different pore sizes differ in composition, life strategies, and activities. Larger pores can better stimulate fast decomposition of newly added C and have a greater abundance of certain microbial taxa compared to smaller pores. The study documents the combined effects of long-term vegetation history on soil pore and POM spatial distribution and hydraulic connectivity in small and large pores. It tests how vegetation history affects bacterial richness, community composition, and metabolism in small and large pores, with consequent effects on soil C processing. Three vegetation systems were tested: a multiyear fallow followed by monoculture corn, a perennial switchgrass community, and a polyculture restored prairie community. μCT was used to characterize soil pore structure and localize POM, with triple-energy μCT examining pore-level water distribution patterns. Labile substrate additions were simulated using labeled glucose, and SIP was used to identify microorganisms assimilating glucose-derived C. Results show that microbial habitats defined by soil pores and POM differ in ways that strongly influence microbial composition and activity, impacting ecosystem processes like decomposition, nitrogen processing, and carbon sequestration. The study highlights the importance of pore-scale hydraulic connectivity in influencing bacterial access to organic matter and subsequent metabolism, with implications for soil carbon accretion and stability. The findings suggest that microbial habitats differ in ecological C-acquisition strategies, with distinct functional roles