Postnatal microbial colonization influences the development of the hypothalamic–pituitary–adrenal (HPA) stress response in mice. The study compared germfree (GF), specific pathogen free (SPF), and gnotobiotic mice to investigate how early microbial exposure affects HPA function. GF mice exhibited a more pronounced HPA stress response to restraint stress, as evidenced by higher plasma ACTH and corticosterone levels, compared to SPF mice. However, no significant difference was observed in response to ether stress. GF mice also showed reduced brain-derived neurotrophic factor (BDNF) expression in the cortex and hippocampus. Reconstitution with Bifidobacterium infantis reversed the exaggerated HPA response, while monoassociation with enteropathogenic Escherichia coli (EPEC) enhanced it. Early reconstitution with SPF faeces partially corrected the HPA response, but not later reconstitution, indicating that early microbial exposure is crucial for HPA system development. These findings suggest that commensal microbiota play a role in shaping the postnatal development of the HPA stress response. The study highlights the bidirectional communication between the gut and brain, with microbial colonization influencing neural systems that regulate stress responses. The results imply that early microbial exposure is essential for the HPA system to become responsive to inhibitory neural regulation. The study also suggests that microbial signals can be transmitted to the brain through both cytokine-mediated and neural pathways. Overall, the research underscores the importance of the gut microbiota in the development of the HPA axis and its role in stress response regulation.Postnatal microbial colonization influences the development of the hypothalamic–pituitary–adrenal (HPA) stress response in mice. The study compared germfree (GF), specific pathogen free (SPF), and gnotobiotic mice to investigate how early microbial exposure affects HPA function. GF mice exhibited a more pronounced HPA stress response to restraint stress, as evidenced by higher plasma ACTH and corticosterone levels, compared to SPF mice. However, no significant difference was observed in response to ether stress. GF mice also showed reduced brain-derived neurotrophic factor (BDNF) expression in the cortex and hippocampus. Reconstitution with Bifidobacterium infantis reversed the exaggerated HPA response, while monoassociation with enteropathogenic Escherichia coli (EPEC) enhanced it. Early reconstitution with SPF faeces partially corrected the HPA response, but not later reconstitution, indicating that early microbial exposure is crucial for HPA system development. These findings suggest that commensal microbiota play a role in shaping the postnatal development of the HPA stress response. The study highlights the bidirectional communication between the gut and brain, with microbial colonization influencing neural systems that regulate stress responses. The results imply that early microbial exposure is essential for the HPA system to become responsive to inhibitory neural regulation. The study also suggests that microbial signals can be transmitted to the brain through both cytokine-mediated and neural pathways. Overall, the research underscores the importance of the gut microbiota in the development of the HPA axis and its role in stress response regulation.