Several lysosomal cathepsins have been implicated in a number of diseases, from arthritis to cancer. A recent member of the cathepsin family, cathepsin S (Cat S) has been associated with several types of cancer in humans. However, to date, no report has linked Cat S to human hepatocellular carcinoma (HCC). Here, we investigated the expression of Cat S in human normal and HCC livers using immunohistochemistry and Western blot analysis. The results showed that no expression or very low levels of Cat S expression were detected in the hepatocytes of normal livers. In contrast, a significant increase in Cat S expression was detected in the cancerous hepatocytes in 34 of the total 63 HCC livers (54%; P<0.01). The Cat S-positive rate was significantly higher in the HCC nodule than in the perinodular region (P<0.01). Nevertheless, the Cat S-positive rate in the peri-HCC region was still significantly higher than that in the normal liver tissue (P<0.01). Elevated Cat S expression in HCC was positively correlated with the presence of portal vein tumor thrombus (P<0.01), extra-hepatic metastasis (P<0.05) and the degree of de-differentiation (P<0.01), but was not correlated with age, the presence of hepatitis B virus surface antigen and cirrhosis, the level of serum α-fetoprotein, the number of tumor nodules, the tumor size and the clinical stage (P>0.05). Aberrant overexpression of Cat S in the cancerous hepatocytes may be one of the key events involved in HCC tumorigenesis, invasion and metastasis.