Background: Diabetes mellitus (DM) refers to a series of metabolic disorders, including elevated blood glucose level diseases due to insufficient insulin secretion or insulin resistance. Objective: To investigate the effect and protective mechanism of muscle-derived stem cell exosomes (MDSC-Exo) on glucolipotoxicity-induced pancreatic β cell injury. Methods: Primary rat muscle-derived stem cells (MDSCs) were isolated and cultured. After the completion of the third-generation culture for MDSCs, MDSC-Exo was isolated. Then, the morphology and diameter of exosomes were observed by means of electron microscopy and nanoparticle tracking analysis (NTA) instrument. The expression of exosome-related proteins CD63, TSG101 and Calnexin was detected by western blot. After stimulation of rat insulinoma cell line INS- 1 with high glucose/palmitic acid (HG/PA) and/or MDSC-Exo, cell viability and apoptosis were measured through MTT and flow cytometry (FCT), respectively. Biochemical reagents were utilized for the examination of the levels of superoxide dismutase (SOD) and malondialdehyde (MDA); enzyme-linked immunosorbent assay (ELISA) for the levels of cellular insulin secretion, and the western blot for the expression level of LC3, p62, AKT, p-AKT, mTOR and p-mTOR. Results: MDSC-Exo was successfully isolated and identified, and it was found that MDSC-Exo could reduce HG/PA-induced apoptosis as well as MDA levels in INS-1 cells. Also, MDSC-Exo could significantly increase cell viability, insulin secretion ability within 24 hours and SOD level. Besides, MDSC-Exo was able to significantly increase the LC3-II/I ratio, decrease the expression level of p62, and promote autophagy in the cells. Aside from what has been mentioned, MDSC- Exo showed a significant reduction effect on p-Akt and p-mTOR level as well as p-Akt/Akt and p-mTOR/mTOR ratios. Conclusion: MDSC-Exo can alleviate oxidative stress and enhance autophagy by inhibiting Akt/ mTOR signaling pathway activation. Then, the inhibition of apoptosis and the promotion of insulin secretion can be achieved to alleviate glucolipotoxicity-induced pancreatic β cell injury.