Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.