Defects in interferon (IFN) signaling that result in loss of expression of IFN-inducible proteins are associated with cellular immortalization, an important early event in the development of human cancer. Here we report that loss of IFN-inducible IFI 16 expression in human fibroblasts allows bypass of cellular senescence. We found that levels of IFI 16 mRNA and protein were higher in human old versus young fibroblasts and immortalization of fibroblasts with telomerase resulted in decreased expression of IFI 16. Moreover, overexpression of IFI 16 in immortalized fibroblasts strongly inhibited cell proliferation. Interestingly, knockdown of IFI 16 expression in fibroblasts inhibited p53-mediated transcription, downregulated p21(WAF1) expression, and extended the proliferation potential. Importantly, treatment of immortal cell lines with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in upregulation of IFI 16. Our observations support the idea that increased levels of IFI 16 in older populations of human fibroblasts contribute to cellular senescence.