Diet is a key factor in fulfilling energy requirements, maintaining metabolic homeostasis and promoting health. Diabetes mellitus is a metabolic disorder characterized by hyperglycemia resulting from defective insulin production or secretion from pancreatic β-cells, insulin function or both. The type, amount as well as balance of main macronutrients (proteins, fats and carbohydrates) present in diet are of particular significance in managing the diabetes. However, relatively little is known about the underlying molecular mechanism that how protein rich diet is responsible for functional changes in pancreatic β-cells. Current study was intended to evaluate the effect of high protein diet on body weight, glycaemic control, regeneration of β-cell, production and release of insulin in diabetic rats. High protein diet (45% and 55% fish protein) was administered in alloxan induced diabetic rat model for a period of 28 days. Blood samples were collected for monitoring blood glucose level, lipid profile and other biochemical parameters. Pancreatic tissue samples were collected for histopathological study. The data was subjected to statistical analysis by applying one way ANOVA and DMR. Results revealed that protein rich diet counteracted the reactive oxygen species (ROS) and normalized the elevated levels of blood glucose (198.1±11.5), cholesterol (64.73±2.24) and triglycerides (65.97±4.62) compared to positive control group having serum glucose (606.8±12.3), cholesterol (95.36±4.6) and triglycerides (109.5±3.51) respectively. Histopathological analysis revealed that high protein diet helps in normal regeneration or replenishing of β-cells. The role of calcium and cellular stress pathways are important in underlying mechanism behind the ROS production and its control as low expression levels of Calm-2, Grk-2, Pias-2, Traf-4, Traf-6 and MAPK-8 genes were observed in high protein diet treated groups in comparison to positive control. High protein diet also improved β-cell function and performance by up regulating the expression of INS-1, INS-2 & Pdx-1 genes. In conclusion, results indicated the regenerative and protective potential of high protein diet in alloxan-induced diabetic rat model.