Endothelin-1 (EDN1) is an important regulator of H+ secretion in the mammalian kidney. EDN1 enhances renal tubule H+-ATPase activity, but the underlying mechanism remains unclear. To further elucidate the role of EDN1 in vertebrates' acid-base regulation, the present study used zebrafish as the model to examine the effects of EDN1 and its receptors on transepithelial H+ secretion. Expression of EDN1 and one of its receptors, EDNRAa, was stimulated in zebrafish acclimated to acidic water. A noninvasive scanning ion-selective electrode technique was used to show that edn1 overexpression enhances H+ secretion in embryonic skin at 3 days post fertilization. EDNRAa loss of function significantly decreased EDN1- and acid-induced H+ secretion. Abrogation of EDN1-enhanced H+ secretion by a vacuolar H+-ATPase inhibitor (bafilomycin A1) suggests that EDN1 exerts its action by regulating the H+-ATPase-mediated H+ secretion. EDN1 does not appear to affect H+ secretion through either altering the abundance of H+-ATPase or affecting the cell differentiation of H+-ATPase-rich ionocytes, because the reduction in secretion upon ednraa knockdown was not accompanied by decreased expression of H+-ATPase or reduced H+-ATPase-rich cell density. These findings provide evidence that EDN1 signaling is involved in acid-base regulation in zebrafish and enhance our understanding of EDN1 regulation of transepithelial H+ secretion in vertebrates.