Vertebrates need to maintain extracellular chloride (Cl 2) concentrations to ensure the normal operation of physiological processes; the transition from aquatic to terrestrial environments necessitated the development of sophisticated mechanisms to ensure Cl 2 homeostasis in the face of fluctuating Cl 2 levels. Zebrafish calcitonin gene-related peptide (CGRP), unlike its splice variant calcitonin, does not respond to environmental Ca 2þ levels. This study aimed to test the hypothesis that CGRP is involved in the control of body fluid Cl 2 homeostasis. Acclimation to high-Cl 2 artificial water stimulated the mRNA expression of cgrp and the receptor (crlr1) when compared with low-Cl 2. CGRP knockdown induced upregulation of the Na þ-Cl 2 co-transporter (ncc2b), while overexpression of CGRP resulted in the downregulation of ncc2b mRNA synthesis and a simultaneous decrease in Cl 2 uptake in embryos. Consistent with these findings, knockdown of either cgrp or crlr1 was found to increase the density of NCC2b-expressing cells in embryos. This is the first demonstration that CGRP acts as a hypochloremic hormone through suppressing NCC2b expression and the differentiation of NCC-expressing ionocytes. Eluci-dation of this novel function of CGRP in fish body fluid Cl 2 homeostasis promises to enhance our understanding of the related physiology in vertebrates.