TRPM2 is a Ca(2+)-permeable nonselective cation channel that plays important roles in oxidative stress-mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an IC(50) of pH(o) = 5.3, whereas internal protons inhibit TRPM2 with an IC(50) of pH(i) = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for pH(o) sensitivity. Mutations of these residues reduce single-channel conductance, decrease external Ca(2+) ([Ca(2+)](o)) affinity, and inhibit [Ca(2+)](o)-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local Ca(2+) concentration at the outer vestibule, therefore reducing [Ca(2+)](o) permeation and inhibiting [Ca(2+)](o)-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the S4-S5 linker is responsible for intracellular pH sensitivity. Replacement of Asp(933) by Asn(933) changes the IC(50) from pH(i) = 6.7 to pH(i) = 5.5. Moreover, substitution of Asp(933) with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/Ca(2+) sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions.