Anoctamin 1 (ANO1)/TMEM16A is a Cl<sup>−</sup> channel activated by intracellular Ca<sup>2+</sup> mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca<sup>2+</sup>. Here, we demonstrate that two helices, “reference” and “Ca<sup>2+</sup> sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca<sup>2+</sup>-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca<sup>2+</sup>-dependent activation because neutralization of these charges change the Ca<sup>2+</sup> sensitivity. We now predict that the Ca<sup>2+</sup> sensor helix attaches to the reference helix in the resting state, and as intracellular Ca<sup>2+</sup> rises, Ca<sup>2+</sup> acts on the sensor helix, which repels it from the reference helix. This Ca<sup>2+</sup>-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.