Members of the Gadd45 family play central roles in the cellular response to genotoxic stress and have been implicated in several human cancers, including hepatocellular carcinomas. Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of primary hepatocellular tumors, although the underlying mechanisms are unclear. Here, we show a novel link between diminished Gadd45beta expression and HCV infection. Inhibited Gadd45beta expression was observed in both nontumoral and tumoral tissues from infected individuals, and in cell lines harboring a HCV replicon and the infectious HCV strain JFH1. Decreased Gadd45beta expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame. Mechanistically, hypermethylation of the Gadd45beta promoter in the presence of HCV is responsible for this defect. Diminished Gadd45beta expression leads to aberrant cell cycle arrest and diminished DNA excision repair. Together, these results provide a novel insight into the mechanisms involved in HCV-associated hepatocellular carcinomas, showing that reduced Gadd45beta expression may play a contributory role to this process, and providing evidence that HCV may interfere with epigenetic gene expression by altering promoter methylation.