Neuroscience research has historically exhibited bias towards male subjects, resulting in limited representation of females in preclinical studies. Despite recent policies emphasizing equal sex representation, questions persist regarding optimal housing practices for male and female animals in research facilities. We aimed to investigate the effects of housing male and female mice with (mixed-sex housing room) or without (same-sex housing room) odor cues from conspecifics on behavior, hippocampal structure, molecular plasticity, and sex-dependent biological function. To do so, we housed two inbred mouse strains (C57BL/6J and Balb/c), were housed in same-sex or mixed-sex rooms using open-top cages from weaning to adulthood. We found strain-specific differences in hippocampal volume, particularly pronounced in the dentate gyrus and cornu ammonis 1 of the ventral hippocampus. These structural changes correlated with transcriptional activity of genes crucial for neurotrophin signaling, synaptic plasticity regulation, and emotional behavior, contributing to differences in exploratory behavior in the open field test. Furthermore, housing conditions affected female estrous cyclicity, with females in same-sex conditions displaying suppressed or prolonged estrous cycles, potentially linked to alterations in steroid hormone production. However, the presence or absence of odor cues from conspecifics did not influence aggressive behavior in male mice but impacted spermatogenesis. These findings suggest a complex interaction between housing conditions, mouse genetic background, and neurobehavioral and reproductive outcomes, offering valuable insights into best practices for animal housing and experimental design, especially for studies investigating sex differences in neuroscience research.