Pupfishes (genus Cyprinodon) evolved some of the broadest salinity tolerances of teleost fishes, with some taxa surviving in conditions from freshwater to nearly 160 ppt. In this study, we examined transcriptional dynamics of ion transporters and aquaporins in the gill of the desert Amargosa pupfish (Cyprinodon nevadensis amargosae) during rapid salinity change. Pupfish acclimated to 7.5 ppt were exposed to freshwater (0.3 ppt), seawater (35 ppt), or hypersaline (55 ppt) conditions over 4 h and sampled at these salinities over 14 d. Plasma osmolality and Cl⁻ concentration became elevated 8 h after the start of exposure to 35 or 55 ppt but returned to baseline levels after 14 d. Osmolality recovery was paralleled by increased gill Na⁺/K⁺-ATPase activity and higher relative levels of messenger RNAs (mRNAs) encoding cystic fibrosis transmembrane conductance regulator (cftr) and Na⁺/K⁺/2Cl⁻ cotransporter-1 (nkcc1). Transcripts encoding one Na⁺-HCO₃⁻ cotransporter-1 isoform (nbce1.1) also increased in the gills at higher salinities, while a second isoform (nbce1.2) increased expression in freshwater. Pupfish in freshwater also had lower osmolality and elevated gill mRNAs for Na⁺/H⁺ exchanger isoform-2a (nhe2a) and V-type H⁺-ATPase within 8 h, followed by increases in Na⁺/H⁺ exchanger-3 (nhe3), carbonic anhydrase 2 (ca2), and aquaporin-3 (aqp3) within 1 d. Gill mRNAs for Na⁺/Cl⁻ cotransporter-2 (ncc2) also were elevated 14 d after exposure to 0.3 ppt. These results offer insights into how coordinated transcriptional responses for ion transporters in the gill facilitate reestablishment of osmotic homeostasis after changes in environmental salinity and provide evidence that the teleost gill expresses two Na⁺-HCO₃⁻ cotransporter-1 isoforms with different roles in freshwater and seawater acclimation.