Treatment with mesenchyme stem cells (MSCs) plays a significant role in the therapies of many diseases such as diabetics. Vitamin D plays a significant role in the development of insulin and can increase the insulin action sensitivity of peripheral tissues. Moreover, there is limited research concerning the mechanism of the therapeutic action of MSCs with the combination of vitamin D (vit. D). Therefore, we evaluated the effect of MSC intervention in a diabetic animal model. Diabetes was induced by streptozotocin (STZ) injection at a dose of 50 mg/kg in adult male rats The diabetic rats were injected with MSCs derived from bone marrow (2 × 106 per rat), either alone or in combination with vit. D through the tail vein for four weeks. Serum insulin, glucose, C-peptide, glycosylated hemoglobin, and lipid profile levels were determined. Pancreatic oxidative stress, histology, and electron microscopy were evaluated, and the gene expression of cytokines was assessed by real-time polymerase chain reaction PCR. MSC treatment suppressed pancreatic inflammatory cytokine secretion and oxidative stress in diabetic rats, resulting in improved pancreatic histology and cellular structure, and the complication of hyperglycemia was observed. Engrafted MSCs were found inside degraded pancreatic regions and regulated inflammatory cytokines. Our results demonstrated that treatment with MSCs and vit. D in combination prevented pancreatic injury via antioxidant and immune regulation in diabetic rats, contributing to the prevention of pancreatic dysfunction, improvement of lipid metabolism, and regulation of cytokine gene expression compared with each one separately. All these mechanisms also improved the histological structure of the pancreas based on transmission electron microscopy. The combination of MSCs and vit. D appears to have contributed to a greater improvement in the diabetic pancreatic complication of rats than was observed by each one separately. Therefore, this association can be used as antidiabetic therapy.