RIP-B7.1 transgenic mice express B7.1 costimulatory molecules in pancreatic islets and develop diabetes following treatment with Poly (I:C), a synthetic mimic of viral dsRNA and an agonist of TLR3 and retinoic acid-inducible protein I (RIG-I). We used this mouse model to elucidate the role of toll-like receptors (TLRs) and the intestinal microbiome in diabetes progression. Only ~10% of the wild-type RIP-B7.1 mice and none of the RIP-B7.1 mice homozygous for targeted disruption of TLR9, TLR3, and MyD88 housed under normal specific-pathogen-free conditions developed diabetes following Poly (I:C) administration (n=12/group). However, antibiotic therapy with trimethoprim-sulfamethoxazole (sulfatrim) plus Poly (I:C) administration led to diabetes in 90% and 100% of the wild type and TLR9-deficient mice, respectively (n = 11-12/group, p ≤ 0.0001 compared to water only for both strains). In contrast, none of the TLR3- or MyD88-deficient RIP-B7.1 mice developed hyperglycemia following the same treatment regimen (n = 9/group, p < 0.0001 compared to TLR9-deficient mice for both strains). High-throughput DNA sequencing of bacterial 16S rRNA from fecal samples demonstrated that the bacterial diversity in TLR9-deficient mice treated with sulfatrim plus Poly (I:C) was higher and slightly increased compared to sulfatrim- plus Poly (I:C)-treated TLR3- and MyD88-deficient mice, respectively (n=6-10/group, p = 0.0002 and p = 0.06, respectively). Principal component analysis suggested that TLR9-deficient mice had distinct gut microbiome compared to the diabetes-resistant TLR3- and MyD88-deficient mice. Finally, administration of sulfatrim plus Poly (I:C) induced a 50-fold increase in the relative abundance of the bacterial phylum and genus Actinobacteria and Bifidobacterium, respectively, in TLR9- versus MyD88-deficient mice (p < 0.05). These data suggest that Poly (I:C)-induced TLR activation and antibiotic-induced alterations in the intestinal microbiome are critical determinants in diabetes development.