AbstractAnoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca²⁺-activated Cl⁻ channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca²⁺-binding pocket; the voltage-dependent binding of two intracellular Ca²⁺ ions to the pocket gates the pore. However, in the absence of intracellular Ca²⁺ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca²⁺ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca²⁺-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca²⁺-binding pocket underlines TMEM16A activation in the absence of intracellular Ca²⁺.