Human calcitonin release is promoted by elevated extracellular Ca2+ (Ca2+o) concentration acting, at least in part, via the calcium-sensing receptor (CaSR). The CaSR is positively modulated by L-amino acids, including the aromatic amino acids L-phenylalanine (Phe) and L-tryptophan (Trp). To investigate the effect of L-amino acids on human calcitonin secretion, we selected thyroid TT cells and exposed them to various Ca2+o concentrations in the absence or presence of L-Phe, plasma-like mixtures of L-amino acids, or the clinically effective positive modulator (calcimimetic) cinacalcet. In the presence of L-Phe or plasma-like mixtures of amino acids, TT cells exhibited enhanced Ca2+o sensitivity in assays of calcitonin release and intracellular Ca2+ mobilization. Furthermore, the effect of elevated Ca2+o and L-Phe on calcitonin release was markedly suppressed by the calcilytic NPS-2143. These effects were dependent on CaSR-mediated activation of Gq/11 as revealed by the specific inhibitor YM-254890. The findings support the hypothesis that calcitonin release is stimulated by increases in plasma L-amino acid levels as well as elevated Ca2+o concentration. They also demonstrate that stimulated calcitonin release as well as basal levels of calcitonin secretion are mediated by a CaSR:Gq/11 signaling mechanism.