The Hedgehog (Hh) pathway has been confirmed a contributor to the carcinogenesis and progression of various tumor types. To investigate the Hh signaling activity in human hepatocellular carcinoma (HCC), we detected the expression levels of Hh pathway components (Shh, Ptch1 and Gli2) in 57 samples of HCC and corresponding adjacent-tumor liver tissues. The Hh pathway was overexpressed in cancer tissues compared with non-cancer tissues and correlated closely with histologic differentiation and portal venous invasion of HCC. To elucidate the relationship between Hh signaling and HCC progression, we performed a further study in vitro. First, the expression levels of the signaling were detected in a subset of hepatoma cell lines and SMMC-7721 cells selected with high level of Hh signaling expression. Next, we employed KAAD-cyclopamine (a specific inhibitor of Hedgehog pathway) to block the Hh pathway in SMMC-7721 cells and assessed the changes of their biological behaviors. The results showed that the blockade of Hh signaling pathway by KAAD-cyclopamine induced reduction of DNA synthesis leading to marked cell growth inhibition and also caused significant attenuation in invasiveness and motility of HCC cells. Collectively, our data demonstrated that the Hh pathway plays an important role in HCC development and invasion. Blockade of the Hh signaling pathway may be a potential target of new therapeutic strategy for HCC.