Diabetes mellitus is a major endocrine disorder and a growing health problem in most countries which can be ameliorated by numerous bio-molecules such as 1α,25 dihydroxyvitamin D3 [1α,25(OH)2VD3]. With this in mind, the current study investigated the therapeutic and preventive effects of 1α,25(OH)2VD3 on diabetes and its side effects: toxicity in liver, pancreas and kidneys. Our results show that administration of 1α,25(OH)2VD3 in diabetic rats increased the plasmatic insulin level, favored the normal blood glucose levels and normalized the hepatic glycogen concentration. In addition, 1α,25(OH)2VD3 enhanced superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) (by 207, 52 and 72%, respectively) compared to diabetic rats. It also reduced lipid peroxidation and the indices of toxicity in liver and kidneys by significantly decreasing alkaline phosphatases (PAL), aspartate and lactate transaminase (AST and ALT) activities, total and direct bilirubin, triglycerides (TG), cholesterol, creatinine, urea and iron levels in diabetic rats. Moreover, the plasmatic non-enzymatic antioxidant level of HDL-cholesterol, magnesium (Mg), calcium (Ca) and copper (Cu) increased after 1α,25(OH)2VD3 administration. The administration of 1α,25(OH)2VD3 in diabetic rats protects against alloxan-induced histological changes in pancreas. Conclusion: from these data, it is concluded that 1α,25(OH)2VD3 might be useful for the therapy and prevention of diabetes and the numerous side effects especially toxicity in liver, pancreas and kidneys and this protective effect is more obvious in our preventive experiment.