Cyanidin-3-O-glucoside (C3G) is a natural colorant with anti-diabetic properties, while its underlying mechanisms remain far from clear. Here, we investigated the protective role of C3G on palmitic acid (PA)-induced pancreatic beta cell dysfunction and further decipher its possible molecular mechanisms. Both primary isolated mouse islets and the INS-1E cell were used, and treated with a mixture of PA (0.5 mM) and C3G (12.5 µM, 25 µM, 50 µM) for different durations (12, 24, 48 h). We found that C3G could dose-dependently ameliorate beta cell secretory function and further alleviate cell apoptosis. Mechanistically, the primary role of the PKR-like ER kinase (PERK) endoplasmic reticulum (ER) stress pathway was detected by RNA sequencing, and the PERK-pathway-related protein expression, especially the pro-apoptotic marker C/EBP homologous protein (CHOP) expression, was significantly downregulated by C3G treatment. The critical role of CHOP in mediating the protective effect of C3G was further validated by small interfering RNA. Conclusively, C3G could ameliorate PA-induced pancreatic beta cell dysfunction targeting the CHOP-related ER stress pathway, which might be used as a nutritional intervention for the preservation of beta cell dysfunction in type 2 diabetes mellitus.