AbstractMS‐275 is a novel synthetic benzamide derivative histone deacetylase (HDAC) inhibitor, that has demonstrated antiproliferative activity in a variety of in vitro human cancer cell lines including breast, colon, lung, myeloma, ovary, pancreas, prostate, and leukemia. Currently, little information is available concerning the effects of MS‐275 on liver cancer cells. In the current study, MS‐275 was found to have potent actions against human hepatoma Hep3B cells including inhibition of cell proliferation and induction of apoptosis. MS‐275 selectively up‐regulated a cyclin‐dependent kinase inhibitor, p21WAF1/Cip1 without alteration of p27WAF1. Expression of p21WAF1/Cip1 is considered to play a pivotal role in Hep3B cell growth arrest and induction of apoptosis. Induction of p21WAF1/Cip1 expression was accompanied by an accumulation of acetylated histones H3 and H4 associated specifically with p21WAF1/Cip1 gene. ChIP analysis revealed remarkable alterations in protein components bound to the promoter region of p21WAF1/Cip1 gene in response to MS‐275 treatment. These included the degradation of HDAC1, HDAC3, and c‐Myc, and as well as increased p300 and RNA polymerase II. The selective effect of MS‐275 on the up‐regulation of the p21WAF1/Cip1 gene whose expression was suppressed in the hepatoma cancer cell line indicated that it would be a very attractive approach in clinical liver cancer therapy. Drug Dev Res 68:61–70, 2007. © 2007 Wiley‐Liss, Inc.