<p>Caffeine is a radio and chemosensitizer; however, its role in chemoprevention remains unknown. Arsenic is a potent carcinogen that threatens human health. Here, we investigated the effects of caffeine on arsenite (NaAsO₂)-induced carcinogenesis. Cytotoxicity was assessed by 2,5-diphenyl-2H-tetrazolium bromide assay and confirmed by morphological observation and fluorescence staining. Cell cycle progression- and apoptosis-related proteins were analyzed by Western blotting. Nuclear factor erythroid2-related factor 2 (NRF2) overexpression was used to confirm the biological function of heme oxygenase-1 (HO-1). NaAsO₂ treatment of A549 (p53 + / +) cells increased the levels of p-ataxia telangiectasia and Rad3-related, p-Chk1, p-Cdc2^Thr161, and p53, which were reduced by caffeine. Caffeine also induced procaspase-3 and HO-1 protein reduction and PARP-1 degradation in a concentration-dependent manner, with similar effects in NaAsO₂-treated cells. The effects of caffeine were reversed by <i>N</i>-acetyl-L-cysteine treatment, which also inhibited JNK and p38 MAPK activation. The increase in HO-1-mediated by NRF2 overexpression inhibited procaspase-3 and PARP-1 degradation and partially upregulated p53 and p21 levels. Treatment with cycloheximide or actinomycin D further reduced caffeine-mediated HO-1, procaspase-3, and PARP-1 degradation. Caffeine increased chemosensitivity to NaAsO₂ by inducing NaAsO₂-induced G1-S phase checkpoint response inhibition and apoptosis through genetic regulation of HO-1. These findings suggest that HO-1 may be an important therapeutic target to overcome NaAsO₂-induced carcinogenesis.</p>