Cathepsin L, a lysosomal acid cysteine protease, was found to be overexpressed in several types of human carcinomas. However, its functional roles in tumor progression and the underlying mechanisms remain largely unclear. In the present study, we investigated a novel functional aspect of cathepsin?L in regulating transforming growth?factor?β (TGF?β)?induced epithelial?mesenchymal transition (EMT) in A549 and MCF?7 cells and examined its possible mechanisms. We found that TGF?β?induced cell morphologic changes of EMT were associated with the increased protein level of cathepsin L in A549 and MCF?7 cells, suggesting that cathepsin L may be involved in the regulation of EMT. Furthermore, we showed that silencing of cathepsin?L blocked TGF?β?induced cell migration, invasion and actin remodeling and inhibited TGF?β?mediated EMT. We also demonstrated that the mechanism of how cathepsin L knockdown regulates EMT may be explained by the suppression of EMT?inducing molecules, such as Snail, which is associated with the phosphatidylinositol 3?kinase (PI3K)?AKT and Wnt signaling pathways. Moreover, we proved that cathepsin?L knockdown in A549 cells significantly inhibited xenograft tumor growth and EMT in?vivo. The results showed a new mechanism to determine cathepsin?L involvement in the regulation of cancer invasion and migration. These results showed that cathepsin L knockdown is important in regulating EMT and suggest that cathepsin L may be utilized as a new target for enhancing the efficacy of chemotherapeutics against epithelial cancer.