The role of TLR in autoimmunity was studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA). Analysis of caecal 16S sequences clearly resolved the mouse lines. The most T1D-susceptible mouse strains had reduced microbiome diversity; about half of sequences from NOD.Tlr1-/- and NOD.Tlr4-/- mice were from Helicobacter and Bacteroides species respectively. Microarray gene expression comparisons of WT NOD, NOD.Tlr1-/- and NOD.Tlr2-/- mice identified highly significant differential expression in multiple pathways affecting the differentiation and integrity of the intestinal mucosa.