Objective To establish interferon-induced transmembrane protein 3 (Ifitm3) knockout mice for exploring the effects of Ifitm3 on the proliferation and differentiation of mouse neural stem cells. Methods The Ifitm3 knockout mice were established by CRISPR/Cas9 method and identified by PCR and Western Blot. The phenotypic differences between the Ifitm3 knockout mice and wild-type mice were analyzed by immunohistochemistry and flow cytometry. Adult mouse neural stem cells were isolated and cultured, the number and size of neurospheres were detected, and markers of proliferation and differentiation of neural stem cells were detected by RT-qPCR, Western Blot and immunofluorescence. Results Ifitm3 knockout mice were successfully established, and the Ifitm3 knockout mice developed normally, and there were no obvious abnormalities in histopathology and immune system. In vitro experiments showed that Ifitm3 affected the self-renewal potential of mouse neural stem cells, led to a decrease in the proliferation ability of neural stem cells, and inhibited the differentiation of neural stem cells into immature neurons and end-stage astrocytes. Conclusion Ifitm3 knockout mice were established using CRISPR/Cas9 method, and neural stem cells were isolated and cultured in vitro, which proved that the lack of Ifitm3 had the effect on the self-renewal, proliferation and differentiation of neural stem cells, providing insights into the biological function of IFITM3.