Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline-to-freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion-motive enzyme activity (V-type H+ ATPase, Na+/K+-ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V-type H+ ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na+/K+-ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V-type H+ ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V-type H+ ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild.