Although both arsenic trioxide (As2O3) and benzo(a)pyrene (BaP) are well-established human carcinogens, the interaction between As2O3 and BaP is synergistic or antagonistic remains controversial in terms of the existing studies. In addition, the mechanisms responsible for the combined effects are still unclear. In this study, we examined the potential interactive effects between As2O3 (1, 5, and 10 μM) and BaP (5, 10, and 20 μM) in cultured A549 cells by treating with BaP and As2O3 alone or in combination at various concentrations for 24 h. The single and combined effects of As2O3 and BaP on the cytotoxicity, DNA/chromosomal damage, and oxidative stress were examined by using tetrazolium (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye colorimetric assay, colony formation assay, fluorescence probe, chemical colorimetry, comet assay as well as micronucleus test. Our results showed that As2O3 synergistically enhanced the cytotoxicity, genotoxicity, and level of oxidative stress induced by BaP at various tested concentrations. Also, our experimental results showed that intracellular glutathione (GSH) contents were increased by various doses of BaP, but single or cotreatment with As2O3 significantly decreased the GSH level in the cells at all tested concentrations. Taken together, our results suggest that As2O3 may exert its synergistic cyto- and genotoxic effects with BaP mainly via elevated intracellular reactive oxygen species and reduced GSH contents and superoxide dismutase activities, thus promoting high level of oxidative stress, which may be a pivotal mechanism underlying As2O3 cocarcinogenic action.