OBJECTIVE: Viral infections contribute to the pathogenesis of type 1 diabetes. Viruses, or viral products such as double-stranded RNA (dsRNA), affect pancreatic beta-cell survival and trigger autoimmunity by unknown mechanisms. We presently investigated the mediators and downstream effectors of dsRNA-induced beta-cell death. RESEARCH DESIGN AND METHODS: Primary rat beta-cells and islet cells from wild-type, toll-like receptor (TLR) 3, type I interferon receptor (IFNAR1), or interferon regulatory factor (IRF)-3 knockout mice were exposed to external dsRNA (external polyinosinic-polycytidylic acid [PICex]) or were transfected with dsRNA ([PICin]). RESULTS: TLR3 signaling mediated PICex-induced nuclear factor-kappaB (NF-kappaB) and IRF-3 activation and beta-cell apoptosis. PICin activated NF-kappaB and IRF-3 in a TLR3-independent manner, induced eukaryotic initiation factor 2 alpha phosphorylation, and triggered a massive production of interferon (IFN)-beta. This contributed to beta-cell death, as islet cells from IFNAR1(-/-) or IRF-3(-/-) mice were protected against PICin-induced apoptosis. CONCLUSIONS: PICex and PICin trigger beta-cell apoptosis via the TLR3 pathway or IRF-3 signaling, respectively. Execution of PICin-mediated apoptosis depends on autocrine effects of type I IFNs.