Immune dysregulation is a recognized phenomenon during spaceflight, including impaired macrophage differentiation and function. Activated macrophages exist in polarized phenotypes, such as M1 macrophages, which produce primarily pro-inflammatory mediators and M2 macrophages that are involved in anti-inflammatory processes and tissue repair. Effective macrophage polarization processes are vital for generating appropriate immune responses and facilitating recovery on Earth and in spaceflight. To gain deeper insight into macrophage polarization processes in spaceflight, we analyzed open-sourced, GeneLab lung tissue transcriptional datasets (OSD-248) from mice previously flown on the Rodent Research (RR)-6 mission. Mice were euthanized on-board the ISS after a 60-day mission. Preliminary analysis revealed an overall decrease in both M1/M2 biosignatures in spaceflight compared to ground controls. Interestingly, select M2 biosignatures were significantly reduced compared to M1, suggesting deficits in tolerogenic/anti-inflammatory activity and a shift towards pro-inflammatory states. In a ground-based study simulating spaceflight conditions, male and female C57BL/6J mice were exposed to simulated galactic cosmic ray radiation combined with hindlimb unloading and social isolation. To assess M1/M2 predominance in the lung and to test the fidelity of a single cell isolation protocol, total macrophages were isolated from frozen-stored lung tissue two weeks post-irradiation exposure. Cells were positively selected for using the F4/80 biomarker, and lung resident and infiltrating macrophage subtypes (M1 and M2) were characterized by flow cytometry, including F4/80, CD170, Arginase-1 (M2), and iNOS (M1). Future studies using tissues from space-flown RR-20 mice will further validate the definition of M1/M2 macrophages in the lung. In summary, characterizing polarized macrophage populations within the lung microenvironment is crucial for advancing our understanding of immune responses in spaceflight, particularly for lunar missions, where astronaut pulmonary physiology will be challenged by unique lunar environmental soil.