Background: Elevated levels of free fatty acids markedly enhance insulin secretion. However, dietary polyunsaturated fatty supplementation decrease insulin secretion. The effects of different type of fatty acids on cultured pancreatic beta cell remain controversy. Therefore, the specific goal of this study was to confirm the effect of palmitate and docosahexaenoic acid (DHA) on pancreatic β-cells. We measured ATP-sensitive K+ (KATP) channel activity by patch clamp technique. Method : Pancreatic β-cells were isolated from male Sprague-Dawley rats and cultured on the cover glass in the culture media. KATP channel activity of pancreatic β-cells were measured by the cell-attached mode of the patch clamp technique. We treated 30μM of palmitate and DHA dissolved with 3% albumin solution. Results : 30μM of palmitate inhibited KATP channel activity. Moreover, after additions of 5 and 10 mM glucose, additional and dose dependent inhibitory effects were revealed. However, 30μM of DHA did not have these additional inhibitory effect treated with 5 and lOmM glucose. Conclusion: Palmitate as a saturated fatty acid inhibited activity of KATP channel and increased inhibitory effect of glucose on this channel activity, however, DHA as a polyunsaturated fatty acid attenuated inhibitory effect of glucose on this channel activity.