||Spaceflight-associated immune system weakening ultimately precludes the expansion of human presence beyond the Earth's orbit. Here, we demonstrate that spaceflight (real) and simulated microgravity significantly reduced the proliferation and differentiation of macrophages and even potentially impaired functional polarization of those macrophages. Using next-generation sequencing (NGS), we find that the genes related cell proliferation and macrophage differentiation were down-regulated while the genes related apoptosis and repair process were up-regulated under microgravity. A systematic analysis of microgravity-caused metabolic alterations indicates that glycolysis is down-regulated and lipid metabolism is up-regulated. Among paramount gene signature, we identify Ras/ERK/NFkB and p53 pathway as the major microgravity-regulated pathways which subsequently impacts survival, proliferation, differentiation, metabolism and following inflammatory effects of macrophage. Quantification of notable genes is validated in mRNA levels and protein levels under simulated microgravity condition. Collectively, our data indicate the relationship of microgravity-response signaling pathways, metabolic reprogramming and macrophage differentiation.