Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.