The ingestion of fat induces secretion of the gut peptide hormone cholecystokinin (CCK); however, the mechanism responsible for lipid-induced CCK release remains unknown. Recently, a group of free fatty acid (FFA) receptors, which includes the long-chain FFA receptors GPR120 and GPR40, has been identified. In this study, we examined whether these FFA receptors mediate lipid-induced CCK release in the mouse. We first observed that intra-gastric administration of long-chain FFAs increased plasma CCK levels. Using mouse enteroendocrine STC-1 cells as a model system, we further studied the mechanism of this FFA-induced CCK secretion. Long-chain FFAs promoted CCK secretion from STC-1 cells, which was abolished either by removal of extracellular Ca2+ or by the L-type Ca2+ channel blocker nicardipine. Furthermore, this FFA-induced CCK secretion was specifically inhibited by transfection of GPR120-specific, but not GPR40-specific, short hairpin RNA. These results indicate that long-chain FFAs induce CCK secretion through GPR120-coupled Ca2+ signaling.