Peimine is a isosteroidal alkaloid with multiple biological activities and has gained widespread clinical applications. This study was designed to investigate the effects of peimine (PM) on breast cancer (BC) and the underlying mechanism. Cell counting kit-8, EdU and transwell migration assays were performed to assess the cell viability, proliferation, and migration of MCF-7 and MDA-MB-231 cells. The interaction between USP41 and O-linked N-acetylglucosamine transferase (OGT) was evaluated by co-immunoprecipitation assay. A xenograft mouse model was established. Results showed that the cell viability of MCF-7 and MDA-MB-231 cells was decreased with the increasing concentration of PM, and the concentration of 20 μM was chosen for followed experiments. Besides, PM suppressed the proliferation and migration of MCF-7 and MDA-MB-231 cells. Moreover, PM treatment decreased the O-linked N-acetylglucosaminylation (O-GlcNAcylation) and OGT protein levels in MCF-7 and MDA-MB-231 cells. Mechanically, USP41 interacted with OGT in MDA-MB-231 cells. Overexpression of OGT enhanced the protein stability of USP41. Final rescue results demonstrated that overexpressing OGT or USP41 reversed the decreases of cell viability, proliferation, and migration in PM-treated MCF-7 and MDA-MB-231 cells
while OGT or USP41 knockout showed opposite results. Animal studies showed that PM treatment inhibited the tumor growth. In summary, PM inhibited cell viability, proliferation, and migration of BC by regulating the O-GlcNAcylation of USP41.