Abstract: In this study, using K562 cells induced by hemin as erythroid differentiation models, the synergistic effects and mechanisms of radiation and simulated microgravity on erythroid differentiation were investigated. Results showed that the positive rates of benzidine staining and the expression of CD235a were down-regulated after treatment with X-ray radiation and simulated microgravity, meanwhile, cell proliferation was inhibited and the rates of apoptosis and necrosis were increased. The expressions of transcription factors related to erythroid differentiation EPOR and GATA-1 were down-regulated. The synergistic effects of X-ray irradiation and simulated microgravity were much stronger than those of the two alone. After irradiation and microgravity combined treatment, PIK3R2 gene expression was down-regulated. After adding PI3K inhibitor 3-MA, the apoptosis and necrosis rate of cells increased, and the erythroid differentiation rates decreased. These results indicate that X-ray irradiation and simulated microgravity treatment have synergistic inhibitory effects on erythroid differentiation, and the mechanism is related to erythroid related transcription factors EPOR, GATA-1 and damage repair pathway factor PI3K.