Promoter hypermethylation of the runt-related transcription factor 3 (RUNX3) gene is associated withincreased risk of hepatocellular carcinoma (HCC). Oxidative stress plays a vital role in both carcinogenesis andprogression of HCC. However, whether oxidative stress and RUNX3 hypermethylation in HCC have a causeand-effect relationship is not known. In this study, plasma protein carbonyl and total antioxidant capacity (TAC)in patients with hepatitis B virus (HBV)-associated HCC (n=60) and age-matched healthy subjects (n=80) wasdetermined. RUNX3 methylation in peripheral blood mononuclear cells (PBMC) of subjects was measured bymethylation-specific PCR. Effect of reactive oxygen species (ROS) on induction of RUNX3 hypermethylationin HCC cells was investigated. Plasma protein carbonyl content was significantly higher, whereas plasma TACwas significantly lower, in HCC patients than healthy controls. Based on logistic regression, increased plasmaprotein carbonyl and decreased plasma TAC were independently associated with increased risk for HCC. PBMCRUNX3 methylation in the patient group was significantly greater than in the healthy group. RUNX3 methylationin hydrogen peroxide (H2O2)-treated HepG2 cells was significantly higher than in untreated control cells. Inconclusion, increase in oxidative stress in Thai patients with HBV-associated HCC was demonstrated. Thisoxidative increment was independently associated with an increased risk for HCC development. RUNX3 in PBMCwas found to be hypermethylated in the HCC patients. In vitro, RUNX3 hypermethylation was experimentallyinduced by H2O2. Our findings suggest that oxidative stress is a cause of RUNX3 promoter hypermethylation inHCC cells.