Background and Aims: Hepatitis B virus (HBV) infec-tion has been found to increase hepatocellular sensitivity to carcinogenic xenobiotics, by unknown mechanisms, in the generation of hepatocellular carcinoma. The pregnane X receptor (PXR) is a key regulator of the body\'s defense against xenobiotics, including xenobiotic carcinogens and clinical drugs. In this study, we aimed to investigate the molecular mechanisms of HBV X protein (HBx)-PXR sign-aling in the synergistic effects of chemical carcinogens in HBV-associated hepatocarcinogenesis. Methods: The ex-pression profile of PXR-cytochrome p4503A4 (CYP3A4) signaling was determined by PCR, western blotting, and tissue microarray. Cell viability and aflatoxin B1 (AFB1) cytotoxicity were measured using the cell counting kit-8 assay. Target gene expression was evaluated using tran-sient transfection and real time-PCR. The genotoxicity of AFB1 was assessed in newborn mice with a single dose of AFB1. Results: HBx enhanced the hepatotoxicity of AFB1 by activating CYP3A4 and reducing glutathione S-transferase Mu 1 (GSTM1) in cell lines. Activation of PXR by pregnenolone 16α-carbonitrile increased AFB1-induced liver tumor incidence by up-regulating oncogenic KRAS to enhance interleukin (IL)-11:IL-11 receptor subunit alpha-1 (IL11RA-1)-mediated inflammation in an HBx transgenic model. Conclusions: Our finding regarding AFB1 toxic-ity enhancement by an HBx-PXR-CYP3A4/ GSTM1-KRAS-IL11:IL11RA signaling axis provides a rational explanation for the synergistic effects of chemical carcinogens in HBV infection-associated hepatocarcinogenesis.