Background: Many variables can lead to one of the most common chronic diseases called type 2 diabetes mellitus. Vitamins K and D are crucial in managing glycemic balance. Objective: This study aimed to evaluate the vitamin K and D supplementation effects on albino rats with streptozotocin-induced type 2 diabetes mellitus, particularly those on a high-fat diet. Material and methods: 78 male albino rats, divided into 6 equal groups. Group I: Normal control group. The remaining 65 adult male albino rats were kept on a high-fat diet for 2 weeks, then injected with streptozotocin for induction of T2DM. Then, they were divided into 5 equal groups. Group II:Diabetic control group, group III: Diabetic taking glimepiride. Group IV: Diabetic taking glimepiride and vitamin D. Group V: Diabetic taking glimepiride and vitamin K. Group VI: Diabetic taking glimepiride, vitamin k and D. After that, blood samples had been collected for estimation of blood glucose, insulin, HOMA IR, vitamin D and vitamin K. Also, gene expression of TGF β, SMAD3 and PDX1 were assessed by PCR. Results: Supplementation with vitamin K or vitamin D3 improved the blood level of glucose, insulin levels, and HOMA-IR compared to the diabetic group. By gene studies the combined-treated group showed a statistically significant decrease in TGF-<0xCE><0xB2> and SMAD3 when compared to other groups, while a statistically significant rise in PDX1 expression was observed. Conclusion: Vitamin K and D supplementation can lower hyperglycemia and hyperinsulinemia in rats with T2DM.