TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca²⁺. This channel contains two independent pores and closely apposed Ca²⁺ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino C. et. al., 2017). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca²⁺ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca²⁺ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca²⁺ binding to enable an otherwise sub-conductive pore to conduct with full capacity.