GW9508 is an agonist of G protein-coupled receptor 40 (GPR40) that is expressed in pancreatic b-cells and is reported to regulate insulin secretion. However, the effects of GW9508 on pancreatic b-cells in primary culture have not been well investigated. This study measured the acute effects of GW9508 on insulin secretion from rat pancreatic islets in primary culture, and the insulin secretion-related events such as the changes in membrane potential, ATP-sensitive potassium currents (K ATP currents), and intracellular Ca 2C concentrations ([Ca 2C ] i ) of rat islet b-cells were also recorded. GW9508 (10-40 mM) did not influence basal insulin levels at 2 mM glucose, but it (above 20 mM) significantly inhibited 5 and 15 mM glucose-stimulated insulin secretion (GSIS). GW9508 did not inhibit insulin secretion stimulated by tolbutamide, the closer of K ATP channels. GW9508 activated K ATP channels and blocked the membrane depolarization and the increase in [Ca 2C ] i that were stimulated by glucose. GW9508 itself stimulated a transient increase in [Ca 2C ] i , which was fully blocked by depletion of intracellular Ca 2C stores with thapsigargin or by inhibition of phospholipase C (PLC) activity with U73122. GW9508-induced activation of K ATP channels was only partly inhibited by U73122 treatment. In conclusion, although it stimulates a transient release of Ca 2C from intracellular Ca 2C stores via activation of PLC, GW9508inhibits GSIS by activating K ATP channels probably in a distal step to GPR40 activation in rat b-cells.