Effective therapies for advanced stages of hepatocellular carcinoma (HCC) have yet to be developed. We investigated how far a combination of the HDAC inhibitor MS-275 and the CDK inhibitor CYC-202 synergizes to inhibit proliferation and promotes apoptosis of hepatoma cells in vitro. Human hepatoma cell lines Hep3B and HepG2 as well as primary human foreskin fibroblasts as non-malignant controls were cultured under standardized conditions and incubated with increasing concentrations of CYC-202 and MS-275 as single agents and in combination. After 24 to 72 h, apoptosis was analyzed by flow cytometry (propidium iodide, JC-1) and by immunocytochemistry for cytokeratin 18 fragmentation. DNA synthesis was assessed using bromodeoxyuridine incorporation. Protein was separated for Western blotting against p21, bax and bcl-2 and fluorimetric activity assays against caspase 3 and 8. The results showed that the combination of CYC-202 and MS-275 leads to better pro-apoptotic effects than the employment of single substances. Apoptosis was induced via the mitochondrial pathway as evidenced by a shift in the bax/bcl-2 ratio and breakdown of mitochondrial transmembrane potentials. Caspase assays revealed a strong induction of caspase 3 but not of the extrinsic initiator caspase 8. In conclusion, combination therapy with the biomodulators MS-275 and CYC-202 is a promising treatment option for HCC.