To investigate the effect of mulberry leaf flavonoids (MLF) on apoptosis of pancreatic cells induced by high glucose. Long exposure to high glucose induces apoptosis of pancreatic β cells, which can lead to diabetes. In this study, we used the rat insulinoma cell line, INS-1. High glucose (33.3 mM) was used to establish a glucotoxicity model. The MTT assay was used to evaluate the MLF effect on cell viability. INS-1 cells were treated with various concentrations of MLF (125, 250 and 500 mg/L) for 24 h, and then stimulated with 5.5 or 33.3 mM glucose for 48 h. Then, the cell supernatants were collected for enzyme-linked immunosorbent assay to determine the level of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor a (TNF-α) and interleukin 6 (IL-6). Western blotting was used to determine the expression of Bcl-2, Bax, caspase-3 and Caspase-9. Cell apoptosis was measured by Annexin V-FITC/propidium iodide double staining and flow cytometry. MLF (125-500 mg/L) improved cell viability. Furthermore, MLF (250 and 500 mg/L) inhibited apoptosis induced by high glucose. The anti-apoptosis effect of MLF was associated with increased SOD, CAT and GSH-Px expression, as well as reduced MDA levels in high-glucose-treated INS-1 cells. Moreover, MLF upregulated Bcl-2 expression, downregulated Bax expression, and reduced the expression of caspase-3 and Caspase-9. Finally, MLF decreased the secretion of inflammatory cytokines and insulin in high-glucose-induced INS-1 cells. MLF is a potential therapeutic agent for preventing diabetes and related disorders.