H⁺-ATPase-rich (HR) cells in zebrafish are known to be involved in acid secretion and Na⁺ uptake mechanisms in zebrafish gills/skin; however, little is known about how HR cells are functionally regulated. In the present work, we studied the roles of Drosophila glial cell missing (gcm), a cell fate-related transcription factor, in the differentiation and functional regulation of zebrafish HR cells. Zebrafish gcm-2 (zgcm2) was found to begin expression in zebrafish embryos at 10 h post-fertilization (hpf), and to be extensively expressed in gills but only mildly so in eyes, heart, muscles, and testes. By whole-mount in situ hybridization, zgcm2 messenger (m)RNA signals were found in a group of cells on the zebrafish yolk-sac surface initially in the tail-bud stage (10 hpf); they had disappeared at 36 hpf, and thereafter appeared again in the gill region from 48 hpf. Double fluorescence in situ hybridization further demonstrated specific co-localization of zgcm2 mRNA in HR cells in zebrafish embryons. Knockdown of zgcm2 with a specific morpholino oligonucleotide caused the complete disappearance of HR cells with a concomitant decrease in H⁺ activity at the apical surface of HR cells, but it did not affect the occurrence of Na⁺-K⁺-ATPase-rich (NaR)cells. A decrease in zatp6v1a (the H⁺-ATPase subunit A) expression and no change in zgcm2 expression in zebrafish gills were seen from 12 h to 3 d after transfer to acidic fresh water, but a compensatory stimulation in the expressions of both genes appeared 4 d post transfer. In conclusion, functional regulation of HR cells is probably achieved by enhancing cell differentiation via zGCM2 activation.