Type I IFNs are known to inhibit tumor cell growth and stimulate the immune system. However, little is known of the mechanism of type I IFN-induced apoptosis in human cancer cells. In this study, we have IFN-beta treatment of a human colorectal cell line (KM12L4) and a resistant clone of this cell line, L4RIFN. We demonstrate the induction of apoptosis in the parent cell line. This process was associated with the induction of the Jak-Stat signaling pathway, induction of the proapoptotic mediator tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activation of procaspase-3, -8, -9, and -10. Additionally, we evaluated the role of Stat1 in mediating IFN-beta induction of these proapoptotic signals in a fibrosarcoma cell line (2ftgh) and a Stat1-deficient clone (U3A). Our results demonstrate that IFN-beta induction of apoptosis and the induction of proapoptotic mediator TRAIL is Stat1 dependent. Evaluation of a stable transfectant of the KM12L4 cell line expressing c-FLIP supports the role of TRAIL and the cell-surface death signaling pathways in IFN-beta induction of apoptosis. Studies evaluating the TRAIL promoter indicate induction of TRAIL promoter activity by IFN-beta. These results may represent a novel pathway by which IFN-beta may induce therapeutic effects.