Background & Aims: Southern Guangxi area is one of the endemic areas for hepatocellular carcinoma (HCC) in China. This study evaluates the roles of genetic variations of hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure in the formation of HCC in this high-risk area. Methods: The study recruited 60 HCC patients and 120 age-, gender-, and residency-matched controls. HBV genotype and basic core promoter (BCP) mutations were determined by nested-PCR/direct sequencing. Serum AFB1-lysine adduct was measured by high performance liquid chromatography-fluorescence detection. Results: HBV genotype C was predominant in 75.0% of cases and 84.2% of controls. The 1762T/1764A double mutations, 1753 V mutations, and 1752V mutations were associated with HCC risk evidenced by the adjusted odds ratio (OR) [95% confidence interval (95% CI)] of 3.89 (1.40-10.77), 2.87 (1.49-5.49), and 5.96 (1.75-20.25), respectively. The adjusted OR (95% CI) was 6.94 (1.68-27.78) for subjects with 1762T/1764A double mutations and high AFB 1-lysine adduct level; 2.01 (0.24-14.29), for those with only 1762T/1764A double mutations; and 4.26 (1.16-15.38) for those with only high AFB1-lysine adduct level, respectively. The adjusted OR was 5.13 (1.79-14.71) for subjects with 1753V mutations and high AFB1-lysine adduct level; 1.20 (0.47-3.08) for those with only 1753V mutations, and 2.28 (1.01-5.31) for those with high AFB1-lysine adduct level, respectively. Conclusions: These data confirmed the association of BCP mutations with HCC risk and the additive effects of 1762T/1764A double mutations and 1753 V mutations with dietary AFB1 exposure in this high-risk area for HCC. © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.