Objective：To investigate the effects of human sperm-associated antigen 5（SPAG5）on proliferation and invasion of breast cancer cells and its molecular mechanism. Methods：The recombinant SPAG5 knockdown/overexpression plasmids siRNA-SPAG5/OE-SPAG5 were designed and constructed respectively transfected with the lentivirus transfection system to prepare stable cell lines. CCK-8 proliferation assay and nude mouse transplantation tumor experimentwere used to detect the proliferation of breast cancer cells.Transwell experiment was used to detect the migration and invasion ability of breast cancer cells.Changes of JAK2/STAT3 pathway and downstream proteins were detected by qRT-PCR and Western blot.The JAK2/STAT3 pathway was inhibited by WP1066 to detect changes in cell proliferation and invasion. Results：The mRNA and protein levels of the SPAG5 in the si1-MDA-MB-231 group were significantly lower than those in the control group，and the mRNA and protein levels of the SPAG5 in the OE-MCF-12A were significantly higher than those in the control group（P < 0.05）. Compared with the control group，the proliferation，migration and invasion ability of the si1-MDA-MB-231 group was inhibited，while these abilities in the OE-MCF-12A group were enhanced（P < 0.05）. Phosphorylation level and downstream protein expression level of JAK2/STAT3 pathway in si1-MDA-MB-231 group were inhibited（P < 0.05），while phosphorylation level and downstream protein expression level of JAK2/STAT3 pathway in OE-MCF-12A group were increased（P < 0.05）. Conclusion：SPAG5 promotes the proliferation and invasion of breast cancer cells through the JAK2/STAT3 signaling pathway