Spatiotemporal changes in branchial ionocyte distribution were investigated following transfer from seawater (SW) to fresh water (FW) in Japanese seabass. The mRNA expression levels of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/K+/2Cl− cotransporter 1a (NKCC1a) in the gills rapidly decreased after transfer to FW, whereas Na+/H+ exchanger 3 (NHE3) and Na+/Cl− cotransporter 2 (NCC2) expressions were upregulated following the transfer. By quadruple-color whole-mount immunofluorescence staining with anti-Na+/K+-ATPase, anti-NHE3, anti-CFTR and T4 (anti-NKCC1a/NCC2) antibodies, we classified ionocytes into one SW-type and two FW-types; NHE3 cell and NCC2 cell. Time-course observation after transfer revealed an intermediate type between SW-type and FW-type NHE3 ionocytes, suggesting functional plasticity of ionocytes. Finally, on the basis of the ionocyte classification of Japanese seabass, we observed the location of ionocyte subtypes on frozen sections of the gill filaments stained by triple-color immunofluorescence staining. Our observation indicated that SW-type ionocytes transformed into FW-type NHE3 ionocytes and at the same time shifted their distribution from filaments to lamellae. On the other hand, FW-specific NCC2 ionocytes appeared mainly in the filaments. Taken together, these findings indicated that ionocytes originated from undifferentiated cells in the filaments and expanded their distribution to the lamellae during FW acclimation.