Objective To explore the effects of transmembrane protein 72(Tmem72)on the proliferation and differentiation of mouse neural stem cells in vitro. Methods The protein expression levels of Tmem72 in different age groups and brain tissues of wild mice were determined by Western blot. A Tmem72 knockdown plasmid was transiently transfected into N2a cells, and proliferation changes after knockdown were identified by CCK8, RT-qPCR, and Western blot. Proliferation and apoptosis changes were also examined with flow cytometry. Mouse neural stem cells were infected with a packaged lentivirus. The knockdown efficiency and changes to proliferation and differentiation marker expression were determined by RT-qPCR and Western blot. Finally, differentially expressed genes and gene-enriched signaling pathways were analyzed by transcriptome sequencing. Results Tmem72 was expressed in different age groups and the whole brain. Compared with the control group, the expression of Tmem72 in N2a cells and neural stem cells in the Tmem72 knockdown group was significantly down-regulated (P< 0. 05). After Tmem72 knockdown, the proliferation ability and activity of N2a cells were significantly increased(P<0. 05), whereas their apoptosis activity was significantly decreased (P<0. 01). The proliferation ability of neural stem cells did not change significantly after Tmem72 knockdown, but differentiation towards astrocytes and immature neurons was promoted (P< 0. 05). Transcriptome sequencing analysis revealed differential gene enrichment in transsynaptic signaling conditions, regulation of glial cell differentiation, PI3K-Akt and MAPK signaling pathways, and ECM-receptor interactions. Tmem72 knockdown promoted the proliferation and inhibited the apoptosis of N2a cells. Tmem72 knockdown promoted the differentiation of neural stem cells into astrocytes and immature neurons, suggesting that Tmem72 is closely related to neural development.