Cathepsin L (CTSL) is a lysosomal cysteine protease that has been found to be overexpressed in ovarian cancer (OC). The aim of the present study was to investigate the possible involvement of CTSL in the development of OC. In this study, RNA interference with a CTSL small hairpin RNA (CTSL-shRNA), and a plasmid carrying CTSL were used to identify the effects of this enzyme on the regulation of the malignant behavior of OC cells. OV-90 and SKOV3 human ovarian cancer cell lines were selected as cell models in vitro and in vivo. The results showed that downregulation of CTSL significantly inhibits the proliferative and invasive capability of SKOV3 cells, and that upregulation of CTSL in OV-90 cells leads to opposite effects. Compared with parental OC cells, cells in which CTSL was silenced exhibited a reduced capacity to develop into tumors in nude mice, while the growth of tumor xenografts derived from these cells was markedly constrained. In conclusion, the results suggested that CTSL contributes to the proliferation and metastasis of OC, and that CTSL may be a novel molecular target for OC treatment.