Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis and hepatocellular carcinoma. HCV NS5A, one of non-structural proteins of HCV, was suggested to play a role in oncogenic transformation. Since the tumor suppressor p53 is important for preventing neoplastic transformation, we investigated the functional effects of HCV NS5A on p53. In vitro and in vivo coimmunoprecipitation and confocal microscopy were used to determine the interaction of NS5A and p53. HCV NS5A binds directly to p53 and colocalizes p53 in the perinuclear region. NS5A inhibits transcriptional transactivation by p53 in a dose-dependent manner by use of a reporter assay. Down regulation of endogenous p21/waf1 expression, which is activated by p53 in Hep3B cells, by NS5A was demonstrated by using FLAG- and FLAG-NS5A Hep3B stable cell lines. The effect of NS5A on p53-mediated apoptosis was determined by flow cytometry in both NS5A permanently and transiently transfected Hep3B cells with exogenous p53. The p53-induced apoptosis was abrogated by NS5A and the inhibition effect correlates well with the binding ability of NS5A to p53. In addition, HCV NS5A protein interacts with and colocalizes hTAF(II)32, a component of TFIID and an essential coactivator of p53, in vivo. These results suggest that HCV NS5A interacts with and partially sequestrates p53 and hTAF(II)32 in the cytoplasm and suppresses p53-mediated transcriptional transactivation and apoptosis during HCV infection, which may contribute to the hepatocarcinogenesis of HCV infection.