BACKGROUND: Beneficial effects of milk protein on glucose metabolism have been reported. OBJECTIVES: We hypothesized that dietary supplementation with specific milk protein fractions could prevent diabetes and differentially alter tissue gene expression. Therefore, we studied the effects of supplementing the diet with whey isolate, whey hydrolysate, α-lactalbumin, and casein proteins in Zucker Diabetic Fatty rats (ZDF) and normal Wistar rats. A chow diet was included as well. METHODS: Six week old male ZDF (n = 60) and Wistar rats (n = 44) were used in a 13 week study. P-glucose, p-insulin, p-glucagon, HbA1c, totalcholesterol, HDL-cholesterol, and triglycerides were measured. An oral glucose tolerance test (OGTT) was performed. Liver, muscle, and adipose samples were used for RT-PCR. One-way ANOVA and multiple comparison tests were performed. RESULTS: HbA1c increased during intervention, and was significantly lower for all milk protein fractions compared to chow in the ZDF rats (p < 0.05). At week 18, iAUCs during OGTT in the ZDF rats were similar for all milk protein-treated groups and significantly lower than in the chow fed group (p < 0.01). In the chow-fed group of ZDF rats, p-glucagon increased significantly compared to all milk protein fed animals. Total and HDL cholesterol were increased in the milk protein-treated ZDF rats compared with the control group. Expression of liver GYS2 and SREBP-2 were downregulated in the milk protein-fed ZDF groups compared with chow. CONCLUSIONS: We conclude that milk protein fractions improve glycemic indices in diabetic rats. No major differences were seen between the milk protein fractions. However, the fractions had a differential impact on tissue gene expression, most pronounced in ZDF rats.