Hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia. HCC is often resistant to chemotherapy and the mechanism remains unclear. Mitochondrion-mediated pathway is critical in hepatocyte apoptosis, which suggests Bcl-2 family genes may play a role in the regulation of chemotherapy in HCC. In the present study, we investigated the role of BH3 domain-only protein Bid in HCC tissues, HCC-derived cell lines and how the expression of Bid was related to chemotherapeutic agent-induced apoptosis. Bid was differently expressed in HCC tissues and hepatoma cell lines. Hep3B, a Bid-abundant HCC cell line, was more sensitive to drug-induced cytotoxicity than PLC/PRF/5, a Bid-insufficient HCC cell line. The level of caspase activity induced by 5-fluorouracil (5-FU) was higher in Hep3B than in PLC/PRF/5 and a significant increase in the activity occurred at a rather late stage, after 48 h of the treatment. Similar to the activation of caspase, Bid cleavage and activation was only significant at 72 h after the treatment. Overexpression of Bid or tBid sensitized HCC cells to 5-FU and doxorubicin (Dox) treatments. We further demonstrated that such a sensitive effect could be offset by Bcl-xL, as Bid- or tBid-induced apoptosis was completely blocked by the over-expression of Bcl-xL. These results indicate the level of Bid expression is closely associated with the sensitivity of HCC cells to chemotherapeutic drugs, suggesting that Bid plays an important role in HCC management.