Free fatty acids (FFAs) stimulate insulin secretion through activation of their receptor, GPR40. It is known that activation of GPR40 leads to an increase in intracellular free Ca2+ concentration ([Ca2+]i), which contributes to the secretion of insulin. Electrophysiological activities of β-cells are crucial in determining levels of [Ca2+]i and insulin secretion, but the action of FFAs on electrophysiological properties of β-cells is largely unknown. Moreover, the mechanism of increase in [Ca2+]i induced by FFAs is not fully understood. We used primary cultured rat pancreatic β-cells to test the effect of linoleic acid on [Ca2+]i and membrane potential. Linoleic acid (20 μM) induced an increase in [Ca2+]i under 3.5 mM glucose, which was eliminated by pretreatment of the cells with thapsigargin, but not blocked by removal of extracellular Ca2+. Simultaneously with the increase in [Ca2+]i, membrane potential was hyperpolarized by linoleic acids significantly (Mean±SD, -48±13.7 mV to -76±6.8 mV after linoleic acids, n=12, P<0.01). Only a very small component of calcium-activated potassium currents was involved, as apamin and charybdotoxin did not deter the hyperpolarization induced by linoleic acid. In contrast, the blockade of ATP-sensitive potassium channels (KATP channels) by tolbutamide totally abolished the hyperpolarization induced by linoleic acid. KATP current was then recorded by nystatin-perorated patch clamp. It was strongly increased by linoleic acid. We concluded that linoleic acid-induced increase in [Ca2+]i is due to calcium release from intracellular calcium stores of rat β-cells but not through voltage-dependent calcium channels. Electrophysiologically, linoleic acid induces hyperpolarization by activating KATP channels, but not calcium-activated potassium channels. This hyperpolarization may prevent insulin secretion induced by a high level of glucose.