Objective To establish interferon-induced transmembrane protein 3 ( Ifitm3) knockout mice and to explore the effects of Ifitm3 on the proliferation and differentiation of adult neural stem cells of mice ( aNSCs). Methods IFITM3 knockout mice were established by the CRISPR/ Cas9 method and identified by genotype identification and Western Blot. The differences between Ifitm3-knockout mice and wild-type mice were analyzed by hematoxylin-eosin(HE) staining and flow cytometry. The aNSCs of wild-type mice and Ifitm3-knockout mice were isolated and cultured, the number and size of neurospheres were detected, The ability of aNSCs to proliferate and differentiate were detected by quantitative reverse-transcription polymerase chain reaction, Western Blot, and immunofluorescence. Results Ifitm3-knockout mice were successfully established. The mice developed normally, and there were no obvious abnormalities either histopathologically or the immune system. In vitro experiments showed that Ifitm3 knockout inhibited the self-renewal potential of aNSCs, led to a decrease in the proliferation ability of aNSCs, and inhibited the differentiation of aNSCs into immature neurons and astrocytes. Conclusions This study finds that a lack of IFITM3 result in the ability of aNSCs to proliferate and differentiate decreased, IFITM3 may regulate the function of aNSCs.