Given NASA’s plans for manned lunar and Mars missions, it is critical to assess the risk of splenic immune dysregulation by using ground-based models of simulated microgravity (SMG) and/or chronic irradiation (CIR). To address this, C57BL/6 J mice of both sexes exposed to SMG and/or CIR for 29 days and alterations in immune cell distribution, function and phenotype were assessed. SMG and/or CIR altered a greater variety of immune cells in both lymphoid and myeloid lineages in female mice than in male mice; the function of splenic CD4 + T cells, CD8 + T cells, and CD19 + B cells altered in a sex-specific manner; and the distribution of different immune cells altered based on animal sex. These findings indicate that SMG and/or CIR alter the splenic immune cell distribution, phenotype and function in a sex-specific manner, underscoring the need for tailored strategies to mitigate health risks for crew members on long-term deep-space missions.