Objective To evaluate the effects of elevated circulating free fatty acids (FFA) level on basal and glucose stimulated insulin secretion (GSIS) of islet β-cell and to explore the pathophysiological link between FFA and impaired β-cell dysfunction. Methods Male SD rats underwent infusions with normal saline (C group), intralipid+heparin (FFA group) and N-acetylcysteine+FFA (NAC group) for 2-4 days. Insulin secretion from pancreatic tissues was evaluated during intravenous glucose tolerance test and isolated pancreas perfasion test at the end of 2 and 4 days infusion. Results After 2 days infusion, the basal insulin secretion from isolated perfused pancreas was increased in FFA group [(55.5±19.4 vs 27.4±6.7) mU/L, P<0.01], but the response to 16.7 mmol/L glucose in isolated perfased pancreas was similar in FFA and C groups. The peak value during GSIS was inhibited by 4 days FFA infusion [(46.8±33.0 vs 214.7±27.4)mIU/L,P<0.05]. GSIS was also decreased in FFA group compared with C group in IVGTr. After interfered with NAC, GSIS was partly recovered [(165.4± 14.8)mIU/L, P<0.01]. Conclusion Elevated circulating FFA levels may contribute to the abnormality of pancreatic islet β-cell through oxidative stress.