It has been well-established that free fatty acids (FFAs) stimulates immediate insulin secretion from pancreatic β-cells, although the underlying mechanism is unclear. An orphan G-protein-coupled receptor, GPR40, has been demonstrated as a specific receptor for FFAs. This receptor is abundantly expressed in pancreatic islet cells. Linoleic acid, an unsaturated FFA, is a high affinity ligand to GPR40. We have shown that linoleic acid significantly decreased the amplitude of voltage-gated K+ currents in primary cultured rat pancreatic β-cells through GPR40 and intracellular cAMP-PKA system. This reduction in K+ currents may indirectly increase Ca2+ influx into and insulin secretion from β-cells. The aim of this study was to clarify the direct effect of linoleic acid on voltage-gated Ca2+ currents in the same cell preparation obtained from rats killed using a CO2 chamber. Nystatin-perforated whole-cell recording configuration was employed to record voltage-gated Ca2+ currents. Using a bath solution containing TEA-Cl (40 mM, K+ channel blocker) and TTX (1μM, Na+ channel blocker) and pipette solution containing Cs+ (to replace K+), two types of Ca2+ currents were isolated by different holding potentials (-80mV and -40mV) and the employment of specific L-type Ca2+ channel blocker (nifedipine) as the L- and T-type Ca2+ currents. The major component of the total Ca2+ currents was the L-type current. Local application of linoleic acid onto recorded cells significantly, and reversibly, decreased the amplitude of both L- and T-type currents, while methyl-linoleate, which has a similar structure to linoleic acid but no binding affinity to GPR40, showed no effect on currents. The linoleic acid-induced reduction in Ca2+ currents was sustained when cells were pre-incubated with a specific protein kinase A inhibitor H89 (1 μM) but was completely abolished by 30 min pre-incubation of cells with a specific intracellular inositol 1,4,5-triphosphate (InsP3) Ca2+ store depleting reagent thapsigargin (1 μM) or 10 min pre-incubation with an inhibitor of the InsP3-gated Ca2+ channel 2-APB (5 μM). We conclude that linoleic acid acts on GPR40 to evoke Ca2+ release from InsP3 sensitive Ca2+ pools, which in turn increases [Ca2+]i levels leading to a reduction of the voltage-gated Ca2+ currents in rat pancreatic β-cells. Supported by NHMRC and Eli Lilly Australia.