Korean pine nut protein (PNP) has a variety of biological activities, which are good for human health, but its ability to preventing diabetes has not been reported. This study evaluated the effects of water-soluble proteins of Korean pine nut obtained from a dilute alkali extract on carbohydrate metabolism of type 2 diabetic mice on a model of diabetes induced using a high fat diet combined with streptozotocin. The results showed that the hypoglycemic effect of PNP at a middle dose was the most significant, which was 38.7% lower than that of control. The extract significantly improved the oral glucose tolerance and liver indexes, increased the activity of the carbohydrate metabolism enzymes, and regulated the expression of the function of key genes for carbohydrate metabolism. It had a positive effect on both insulin resistance and glycolytic/gluconeogenesis signaling. In conclusion, PNP can regulate fasting blood glucose, improve insulin resistance, correct the glucose metabolism disorder in diabetic mice, and have a positive regulatory role. As the functional food, it has the potential to be beneficial in the treatment of type 2 diabetes mellitus as a new hypoglycemic functional food.