TMEM16F, a dual functional phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pH_i). We found that low pH_i attenuates, whereas high pH_i potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca²⁺ ([Ca²⁺]_i). We further demonstrate that TMEM16F pH_i sensitivity depends on [Ca²⁺]_i and exhibits a bell-shaped relationship with [Ca²⁺]_i: TMEM16F channel activation becomes increasingly pH_i sensitive from the resting [Ca²⁺]_i to micromolar [Ca²⁺]_i, but when [Ca²⁺]_i increases beyond 15µM, the pH_i sensitivity gradually diminishes. The mutation of a Ca²⁺-binding residue that markedly reduces TMEM16F Ca²⁺ sensitivity (E667Q), maintains the bell-shaped relationship between pH_i sensitivity and Ca²⁺ but causes a dramatic shift of the peak [Ca²⁺]_i from 15µM to 3 mM. Our biophysical characterizations thus pinpoints that the pH_i regulatory effects on TMEM16F stem from the competition between Ca²⁺ and proton for the primary Ca²⁺-binding residues in the pore. Within the physiological [Ca²⁺]_i range, the protonation state of the primary Ca²⁺-binding sites influences Ca²⁺ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pH_i and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pH_i including cancer.