With advancements in deep space exploration missions, long-term spaceflights pose potential hazards to the reproductive and developmental functions of astronauts. Embryonic stem cells (ESCs), which are crucial to the development and growth of individual organisms, are observably altered by a microgravity environment. However, the role and mechanisms of microgravity in other activities of ESCs are still unclear. Here, mouse embryonic stem cells (mESCs) were used to investigate and understand the effect of microgravity on their activities. Combined with the SJ-10 satellite and random position machine, which were utilized for spaceflight and microgravity simulation, respectively, the bioinformatic tools were also used to assess the effect that microgravity might have on mESC activities. Based on differentially expressed genes (DEGs) analysis, 114 DEGs were significantly up-regulated and 859 DEGs were significantly down-regulated in mESCs after being subjected to spaceflight. The activities, such as cell proliferation, senescence, and apoptosis, were selected and confirmed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. It demonstrated a reduced proliferation capability of mESCs but increased the number of senescent and apoptotic cells after being subjected to simulated microgravity. Real-time polymerase chain reaction analysis of the screened activity-related DEGs demonstrated approximately consistent trends of these gene expressions in both spaceflight and simulated microgravity, as was predicted by bioinformatics analysis. Overall, these findings suggest that microgravity inhibits the proliferation of mESCs and induces senescence and apoptosis, shedding light on the impact of microgravity on the fundamental functions of mESCs in reproductive and embryonic development.