Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.