The sense of taste allows animals to sample chemicals in the environment prior to ingestion. Of the five basic tastes, sour, the taste of acids, had remained among the most mysterious. Acids are detected by type III taste receptor cells (TRCs), located in taste buds across the tongue and palate epithelium. The first step in sour taste transduction is believed to be entry of protons into the cell cytosol, which leads to cytosolic acidification and the generation of action potentials. The proton-selective ion channel Otop1 is expressed in type III TRCs and is a candidate sour receptor. Here, we tested the contribution of Otop1 to taste cell and gustatory nerve responses to acids in mice in which Otop1 was genetically inactivated (Otop1-KO mice). We first show that Otop1 is required for the inward proton current in type III TRCs from different parts of the tongue that are otherwise molecularly heterogeneous. We next show that in type III TRCs from Otop1-KO mice, intracellular pH does not track with extracellular pH and that moderately acidic stimuli do not elicit trains of action potentials, as they do in type III TRCs from wild-type mice. Moreover, gustatory nerve responses in Otop1-KO mice were severely and selectively attenuated for acidic stimuli, including citric acid and HCl. These results establish that the Otop1 proton channel plays a critical role in acid detection in the mouse gustatory system, evidence that it is a bona fide sour taste receptor.