Summary. Background: Human eNOS (NOS3) polymorphisms that lower its expression are associated with advanced diabetic nephropathy (DN), and the lack of eNOS accelerates DN in diabetic mice. Diabetes is associated with fibrin deposition. Lack of nitric oxide and fatty acids stimulates the NF-kB pathway, which increases tissue factor (TF). Objectives: To test the hypothesis that TF contributes to the severity of DN in the diabetic eNOS−/− mice fed a high-fat diet (HF). Methods: We made eNOS−/− and wild-type mice diabetic with streptozotocin. Half of them were placed on HF. Results: Blood glucose levels were not affected by either the diet or eNOS genotype. Lack of eNOS in the diabetic mice increased urinary albumin excretion, glomerulosclerosis, interstitial fibrosis, and glomerular basement membrane thickness. HF by itself did not affect DN in the wild-type mice, but significantly enhanced DN in eNOS−/− mice. More than half of diabetic eNOS−/− mice on HF died prematurely with signs of thrombotic complications. Diabetic kidneys contained fibrin and TF, and their levels were increased by the lack of eNOS and by HF in an additive fashion. The HF diet increased the kidney expression of inflammatory genes. The increase in TF preceded DN, and administration of an anti-mouse TF antibody to diabetic mice reduced the expression of inflammatory genes. Conclusion: Together, these data indicate a causal link between TF and the exacerbation of DN in eNOS−/− mice. The condition is significantly worsened by enhanced inflammatory responses to an HF diet via TF.