Objective To investigate the effect of RBM5 gene knockout by CRISPR/Cas9 system on the biological behavior of endometrial cancer cells. Methods CRISPR/Cas9⁃RBM5⁃sgRNA recombinant plasmids were transfected intoRBM5 ⁃ high expression Ishikawacell lines.Western blot，qRT ⁃ PCR and Sanger sequencing were used to verify the RBM5 knockout. The abilities of proliferation，migration and invasion of parental strains，knock⁃ out strains and revertant strains were analyzed by CCK⁃8 kit，colony formation and Transwell assays. Results The expression of RBM5 was significantly decreased in the knockout cell lines（P < 0.05）. The sequencing results showed a code shift mutation. The abilities of proliferation ，migration and invasion in the RBM5⁃knockout cell significantly increased（P < 0.05）. Conclusion RBM5 knockout in endometrial cancer cell lines can promote cell proliferation，migration and invasion，which guides further molecular mechanism of endometrial cancer.