Abstract: Objective To construct the short hairpin RNA(shRNA) eukaryotic expressing vector targeting human papillomavirus type 16 E6/E7 to establish stable transfected SiHa cell line and to observe its effects on cell proliferation and migration. Methods Three pairs of shRNA fragments which could specifically inhibit HPV16 E6/E7 genes were synthesized and cloned into the shRNA vector psilencer2.1-U6 hygro. The recombinant vectors were transfected into SiHa cells by Lipo2000 and E6/E7 mRNA expression was validated by real-time PCR. The stable psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line was selected with hygromycin B and validated by real-time PCR and Western blot.The CCK-8 cell proliferation assay and cell scratch wound healing assay were performed to examine cell proliferation and migration. Results Sequencing suggested that shRNA eukaryotic expression vector targeting HPV16 E6/E7 gene, psilencer 2.1-U6 hygro-shE6/E7-SiHa cell line, was established successfully with a signifi cantly inhibition of HPV16 E6/E7 expression, cell proliferation and migration. Conclusion Inactivation of HPV16 E6/E7 in cervical cancer cells could reduce cell proliferation and migration.