Transient Receptor Potential Vanilloid 3 (TRPV3) forms Ca2+-permeable nonselective cation channels highly expressed in keratinocytes. Gain-of-function mutations in TRPV3 result in spontaneous dermatitis and pruritus in humans and rodents. TRPV3 also plays important roles in hair morphogenesis and maintenance of epidermal barrier function. Thus, understanding the mechanism of regulation of TRPV3 has important implications in skin health. Previously, it has been shown that protons exert both stimulatory and inhibitory actions on TRPV3. While the large part of the stimulatory action may be attributed to the direct modification by protons of the TRPV3 ligand, 2-aminoethoxydiphenyl borate (2APB), used in these studies, the mechanism of proton inhibition of TRPV3 remains unknown. Here, we examined the extracellular and intracellular effects of low pH on TRPV3 expressed in HEK 293 cells using whole-cell and inside-out patch clamp techniques. Lowering extracellular pH from 7.2 to 6.5 and 5.5 caused a transient and persistent inhibition of TRPV3 whole-cell currents evoked by 2APB and carvacrol, respectively. The inhibition was abolished not only by the mutation of Asp 641 to Asn at the selectivity filter of the channel, but also two glutamate and one aspartate residues at its C-terminus. However, only with the D641N mutation, did intracellular acidification become stimulatory in the absence of any other TRPV3 ligand, suggesting Asp 641 to be the key residue for proton inhibition. Our study reveals a critical residue involved in TRPV3 inhibition by low pH and several protonatable residues at the C-terminus of TRPV3 that regulate the overall functionality of the channel.