To investigate the molecular mechanism of leptin regulating insulin secretion through determining the regulation of insulin secretion and the insulin receptor substrate (IRS)-2-associated phosphoinositide 3-kinase (PI3K) activity by leptin in the isolated rat pancreatic islets, pancreatic islets were isolated from male SD rats by the collagenase method. The purified islets were incubated with leptin 2 nmol/L for 1 h in the presence of 5.6 mmol/L or 11.1 mmol/L glucose. Insulin release was measured using radioimmunoassay. IRS-2-associated activity of PI3K was determined by immunoprecipitate assay and Western blot. The results showed that in the presence of 5.6 mmol/L glucose, leptin had no significant effect on both insulin secretion and IRS-2-associated PI3K activity, but in the presence of 11.1 mmol/L glucose, insulin release was significantly inhibited after the islets were exposed to leptin for 1 h (P<0.01). PI3K inhibitor wortmannin blocked the inhibitory regulation of leptin on insulin release (P<0.05). Western Blot assay revealed that 2 nmol/L leptin could significantly increase the IRS-2-associated activity of PI3K by 51.5 % (P<0.05) in the presence of 11.1 mmol/L glucose. It was concluded that Leptin could significantly inhibit insulin secretion in the presence of 11.1 mmol/L glucose by stimulating IRS-2-associated activity of PI3K, which might be the molecular mechanism of leptin regulating insulin secretion. To investigate the molecular mechanism of leptin regulating insulin secretion through determining the regulation of insulin secretion and the insulin receptor substrate (IRS) -2-associated phosphoinositide 3-kinase (PI3K) activity by leptin in the isolated rat pancreatic islets, pancreatic islets were isolated from male SD rats by the collagenase method. The purified islets were incubated with leptin 2 nmol / L for 1 h in the presence of 5.6 mmol / L or 11.1 mmol / L glucose. Insulin release was measured using radioimmunoassay. IRS-2-associated activity of PI3K was determined by immunoprecipitate assay and Western blot. The results showed that in presence of 5.6 mmol / L glucose, leptin had no significant effect on both insulin secretion and IRS-2-associated PI3K activity, but in presence of 11.1 mmol / L glucose, insulin release was significantly inhibited after the islets were exposed to leptin for 1 h (P <0.01). PI3K inhibitor wortmannin blocked the inhibitory regulation of leptin on in Western Blot assay revealed that 2 nmol / L leptin could significantly increase the IRS-2-associated activity of PI3K by 51.5% (P <0.05) in the presence of 11.1 mmol / L glucose. It was concluded that Leptin could significantly inhibit insulin secretion in the presence of 11.1 mmol / L glucose by stimulating IRS-2-associated activity of PI3K, which might be the molecular mechanism of leptin regulating insulin secretion.