Understanding the mechanisms underlying oro-gustatory detection of dietary fat is critical for the prevention and treatment of obesity. The lipid-binding glycoprotein CD36, which is expressed by circumvallate papillae (CVP) of the mouse tongue, has been implicated in oro-gustatory perception of dietary lipids. Here, we demonstrate that stromal interaction molecule 1 (STIM1), a sensor of Ca(2+) depletion in the endoplasmic reticulum, mediates fatty acid-induced Ca(2+) signaling in the mouse tongue and fat preference. We showed that linoleic acid (LA) induced the production of arachidonic acid (AA) and lysophosphatidylcholine (Lyso-PC) by activating multiple phospholipase A2 isoforms via CD36. This activation triggered Ca(2+) influx in CD36-positive taste bud cells (TBCs) purified from mouse CVP. LA also induced the production of Ca(2+) influx factor (CIF). STIM1 was found to regulate LA-induced CIF production and the opening of multiple store-operated Ca(2+) (SOC) channels. Furthermore, CD36-positive TBCs from Stim1-/- mice failed to release serotonin, and Stim1-/- mice lost the spontaneous preference for fat that was observed in wild-type animals. Our results suggest that fatty acid-induced Ca(2+) signaling, regulated by STIM1 via CD36, might be implicated in oro-gustatory perception of dietary lipids and the spontaneous preference for fat.