Myostatin, a negative modulator of muscle growth, has been considered as a potential target for the treatment of type 2 diabetes (T2D). In previous work, it was found that myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of myostatin propeptide (MPRO) could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in db/db mice. This study investigated whether pre-intervention of rAAV-mediated expression of MPRO could lower the incidence of T2D. Three-week-old male C57BL/6 mice were randomly divided into saline control, rAAV-GFP, and rAAV-MPRO groups, all of which were fed on a high-fat diet. It was observed that pre-intervention of rAAV-MPRO prevented high-fat diet-induced hyperglycemia and hyperlipidemia. It also improved glucose tolerance, downregulated serum insulin levels, and facilitated the growth of skeletal muscle and fat redistribution, with no significant difference in serum free fatty acid levels and body weight, which ultimately reduced the incidence of T2D. In addition, pretreatment of rAAV-MPRO in C2C12 cells increased insulin-stimulated glucose uptake, as well as glycogen synthesis under insulin resistance conditions induced by free fatty acids, with no significant difference in insulin-stimulated glucose oxidation. Finally, the study demonstrated that improved glucose metabolism by rAAV-MPRO pretreatment might be due to the activation of the PI3K/Akt/GSK3 beta pathway and spurring Glut4 transposition from the cytoplasm to the cytomembrane in C2C12 cells. Based on these findings, MPRO is most likely to be a new method for the prevention of T2D.