N-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end-products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic beta cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, effects of alpha-lipoic acid (ALA) on mitochondria in AGEs-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 weeks increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxides production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells.Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 (PINK1) protein suggest that fragmented mitochondria were associated with increased mitophagic activity and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic beta cells. The findings from this study suggest that increased concentration of AGEs may damage beta cells and reduce insulin secretion.