Tumor necrosis factor-α (TNF-α) is an important inflammation cytokine without known direct effect on DNA. In this study, we found that TNF-α can cause DNA damages through reactive oxygen species. The mutagenic effect of TNF-α is comparable with that of ionizing radiation. TNF-α treatment in cultured cells resulted in increased gene mutations, gene amplification, micronuclei formation, and chromosomal instability. Antioxidants significantly reduced TNF-α–induced genetic damage. TNF-α also induced oxidative stress and nucleotide damages in mouse tissues in vivo. Moreover, TNF-α treatment alone led to increased malignant transformation of mouse embryo fibroblasts, which could be partially suppressed by antioxidants. As TNF-α is involved in chronic inflammatory diseases, such as chronic hepatitis, ulcerative colitis, and chronic skin ulcers, and these diseases predispose the patients to cancer development, our results suggest a novel pathway through which TNF-α promotes cancer development through induction of gene mutations, in addition to the previously reported mechanisms, in which nuclear factor-κB activation was implicated. (Cancer Res 2006; 66(24): 11565-70)