The aim of this study was to investigate whether xanthohumol may exert chemoprotective activity through the modulation of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in immortalized normal THLE-2 hepatocytes and a hepatocellular carcinoma HepG2 cell line. Cells were incubated in the presence of xanthohumol and the activation of Nrf2 and expression of genes controlled by this transcription factor were evaluated. Additionally, p53 level was assessed. Xanthohumol increased the expression and led to the activation of Nrf2 in both cell lines. However, in contrast to normal cells the expression of genes controlled by this transcription factor was not affected in HepG2 cells, except for GSTA and GSTP. Xanthohumol, beside the induction of GSTs and HO-1, significantly elevated NQO1 expression in concert with p53 level in normal hepatocytes. The activation of Nrf2 pathway and subsequently phase II enzymes in concert with p53 induction in normal hepatocytes may account for the molecular mechanism of the chemopreventive activity of xanthohumol. On the other hand its cytotoxicity towards HCC cells shown in this study indicates that it may also be considered as potentially chemotherapeutic.