Arsenic has been linked to increased prevalence of cancer and cardiovascular disease (CVD), but the long-term impact of arsenic exposure remains unclear. Human paraoxonase (PON1) is a high-density lipoprotein-associated antioxidant enzyme which hydrolyzes oxidized lipids and is thought to be protective against atherosclerosis, but evidence remains limited to case-control studies. Only recently have genes encoding enzymes responsible for arsenic metabolism, such as AS3MT and GSTO, been cloned and characterized. This study was designed to evaluate the synergistic interaction of genetic factors and arsenic exposure on electrocardiogram abnormality. A total of 216 residents from three tap water implemented villages of previous arseniasis-hyperendemic regions in Taiwan were prospectively followed for an average of 8 years. For each resident, a 12-lead conventional electrocardiogram (ECG) was recorded and coded by Minnesota Code standard criteria. Eight functional polymorphisms of PON1, PON2, AS3MT, GSTO1, and GSTO2 were examined for genetic susceptibility to ECG abnormality. Among 42 incident cases with ECG deterioration identified among 121 baseline-normal subjects, arsenic exposure was significantly correlated with incidence of ECG abnormality. In addition, polymorphisms in two paraoxonase genes were also found associated with the incidence of ECG abnormality. A haplotype R-C-S constituted by polymorphisms of PON1 Q192R, -108C/T and PON2 C311S was linked to the increased risk. Subjects exposed to high levels of As (cumulative As exposure >14.7 ppm-year or drinking artesian well water >21 years) and carrying the R-C-S haplotype had significantly increased risks for ECG abnormality over those with only one risk factor. Results of this study showed a long-term arsenic effect on ECG abnormality and significant gene-gene and gene-environment interactions linked to the incidence of CVD. This finding might have important implications for a novel and potentially useful biomarker of arsenic risk.