Abstract: Objective To investigate whether Cathepsin L affects the invasion and migration of ovarian cancer cells SKOV-3 via regulating epithelial mesenchymal transition(EMT). Methods The expression of Cathepsin L in ES-2, SKOV3, OV1 and OV2 cell lines were analyzed by Real-time PCR. shRNA targeting Cathepsin L was designed and synthesized, and then transfected into SKOV-3 cells which expressed the highest level Cathepsin L in four cell lines via Lipofectamine 2000 mediation. Western blot and Real-time PCR were used to detect the expression of Cathepsin L in SKOV-3 cells which were transfected with shRNA. The migration ability of SKOV-3 was analyzed by Wound scratch assay. The invasion ability was detected by Transwell assay. The expression of E-cadherin, N-cadherin, p-AKT and Snail were detected by Western blot. Results The expression of Cathepsin L mRNA and protein were the highest in SKOV3 cells, while those in ES-2, OV1 and OV2 were (0.72±0.04), (0.34±0.03) and (0.55±0.05). Capthesin L-shRNA could significantly down-regulate Cathepsin L expression in SKOV-3/shRNA. Cell migration ability was significantly inhibited in shRNA interference group at 12 and 24h time points. The migration cells of SKOV3/shRNA was (35.67±4.73), while those of the SKOV3 and SKOV3/Con groups were (93.67±8.62) and (90.33±12.22)(P<0.001).In addition, the expression of E-cadherin was increased, while those of N-cadherin, p-AKT and Snail were decreased in shRNA interference group. Conclusion Cathepsin L gene could promote the invasion and migration of ovarian cancer cells SKOV-3 via regulating upstream signaling pathway of EMT, suggesting that Cathepsin L plays an important role in the development of ovarian cancer.