This study presents high-resolution in situ structures of mammalian respiratory supercomplexes (SCs) using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). The research directly images porcine mitochondria to determine the structures of various high-order assemblies of respiratory SCs in their native states. Four main SC organizations were identified: I₁III₂IV₁, I₁III₂IV₂, I₂III₂IV₂, and I₂III₄IV₂. These SCs are largely formed by 'protein–lipids–protein' interactions, which significantly impact the local geometry of the surrounding membranes. The structures reveal numerous reactive intermediates within these SCs, providing insights into the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparisons of SCs under different conditions suggest a possible correlation between conformational states of complexes I and III, likely in response to environmental changes. The in situ approach preserves the native membrane environment, enabling structural studies of mitochondrial respiratory SCs at high resolution across multiple scales, from atomic-level details to the broader subcellular context. The study also identifies multiple Q-binding states within complex I, revealing distinct conformations of ubiquinone (Q₁₀) in the Q-binding channel. These states include an apo state and three new Q-bound states, which provide insights into the Q₁₀ movement and proton transfer mechanisms. The research further explores the active and deactive states of complex I under physiological conditions, showing that the deactive state may arise with gradual substrate depletion. The structures also reveal the dynamic intermediates of complex II (ClII₂), including the Q-cycle mechanism and the conformational changes in the Rieske domain. The study highlights the conformational correlation between complex I and ClII₂, indicating that the conformational states of these complexes may be interdependent. The findings suggest that the local geometry of the membrane surrounding SCs fits the overall shape of the planar regions of mitochondrial cristae, consistent with previous findings that SCs predominantly localize in these regions. The study also discusses the potential functional roles of SCs, including their role in regulating protein stability, minimizing reactive oxygen species production, and preventing age-associated protein aggregation. The research provides new insights into the mechanisms of mitochondrial SCs that reflect physiological behavior and has potential applications in studying the effects of various compounds on mammalian mitochondria. The in situ imaging approach used in this study could be extended to structural studies of native membrane protein complexes within various organelles.This study presents high-resolution in situ structures of mammalian respiratory supercomplexes (SCs) using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). The research directly images porcine mitochondria to determine the structures of various high-order assemblies of respiratory SCs in their native states. Four main SC organizations were identified: I₁III₂IV₁, I₁III₂IV₂, I₂III₂IV₂, and I₂III₄IV₂. These SCs are largely formed by 'protein–lipids–protein' interactions, which significantly impact the local geometry of the surrounding membranes. The structures reveal numerous reactive intermediates within these SCs, providing insights into the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparisons of SCs under different conditions suggest a possible correlation between conformational states of complexes I and III, likely in response to environmental changes. The in situ approach preserves the native membrane environment, enabling structural studies of mitochondrial respiratory SCs at high resolution across multiple scales, from atomic-level details to the broader subcellular context. The study also identifies multiple Q-binding states within complex I, revealing distinct conformations of ubiquinone (Q₁₀) in the Q-binding channel. These states include an apo state and three new Q-bound states, which provide insights into the Q₁₀ movement and proton transfer mechanisms. The research further explores the active and deactive states of complex I under physiological conditions, showing that the deactive state may arise with gradual substrate depletion. The structures also reveal the dynamic intermediates of complex II (ClII₂), including the Q-cycle mechanism and the conformational changes in the Rieske domain. The study highlights the conformational correlation between complex I and ClII₂, indicating that the conformational states of these complexes may be interdependent. The findings suggest that the local geometry of the membrane surrounding SCs fits the overall shape of the planar regions of mitochondrial cristae, consistent with previous findings that SCs predominantly localize in these regions. The study also discusses the potential functional roles of SCs, including their role in regulating protein stability, minimizing reactive oxygen species production, and preventing age-associated protein aggregation. The research provides new insights into the mechanisms of mitochondrial SCs that reflect physiological behavior and has potential applications in studying the effects of various compounds on mammalian mitochondria. The in situ imaging approach used in this study could be extended to structural studies of native membrane protein complexes within various organelles.