The aim of this study was to investigate the protective effects of a chokeberry anthocyanin extract (CAE) on pancreaticβ-cells (βTC3) exposed to hydrogen peroxide- (H₂O₂-) and high glucose- (HG-) induced oxidative stress conditions. In order to quantify individual anthocyanins high performance liquid chromatography (HPLC) coupled to photodiode array (PDA) was used. The identification of the fragment ion pattern of anthocyanins was carried out by electrospray ionization mass spectrometry (LC-ESI-MS). The results showed that physiologically achievable concentrations of CAE (1, 5, and 10 μM) protectβTC3 against H₂O₂- and HG-induced cytotoxicity. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were increased in pancreaticβ-cells pretreated with CAE compared to cells exposed to the prooxidant agents. GSH levels initially reduced after exposure to H₂O₂and HG were restored by pretreatment with CAE. Insulin secretion inβTC3 cells was enhanced by CAE pretreatment. CAE restored the insulin pool and diminished the intracellular reactive oxygen species level in glucose-induced stress condition inβTC3 cells. These results demonstrate that anthocyanins from CAE were biologically active, showing a secretagogue potential and an antioxidative protection of enzymatic systems, conferring protection against H₂O₂and glucose toxicity inβTC3 cells.