Acid-sensing ion channel (ASIC) has a prominent role in tissue pH-sensing and neuronal signaling, and participates in various pathological conditions.Despite the availability of structures for chicken ASIC1 subunit, obtaining mechanistic insight into the function of these highly dynamic channel proteins using static crystallographic information is challenging.Using a series of mutational, chemical and functional approaches, we found that the extracellular Cl--binding module shown in crystals of ASIC1 constitutes an indispensable component for ASIC1a function.Massive gluconate-substitution of natural Cl--binding enhanced whereas engineering a permanent anion into the Cl--binding module inhibited channel activity, suggesting an essential role of Cl--regulated domain flexibility but not the absolute charge state created by bound anion in channel operation.Size-comparable anion substitution (F, Br, or I-) revealed a Cl--dependent modulation of proton sensing function, supporting a structural role of dynamic association and dissociation of Cl-ion in coordinating conformational transition associated with proton sensing.Furthermore, charge reversal and swap as well as mutant cycle analysis of alanine substitutions of two counter-charged residues (K311, E315) in the Cl--binding module show that they electrostatically interacted and energetically coupled, underpinning a structural switch controlling proton sensitivity of ASIC1a channels.