Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance, to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavorable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs-the gills and intestine. Using immunofluorescence, we localized aquaporin-3 (AQP3) protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial AQP3 messenger-RNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter AQP3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish AQP3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater and facilitates intestinal permeability in seawater and freshwater.