The morphology of fish gills is closely linked to aerobic capacity and tolerance of environmental stressors such as hypoxia. The importance of gill surface area is well studied, but little is known about how the mechanical properties of gill tissues determine function. In some fishes, the bases of the gill filaments are surrounded by a calcified ‘sheath' of unknown function. We tested two non-exclusive hypotheses: (i) calcified gill filaments enhance water flow through the gill basket, improving aquatic respiratory function, and (ii) in amphibious fishes, calcification provides support for gills out of water. In a survey of more than 100 species of killifishes and related orders, we found filament calcification was widespread and thus probably arose before the evolution of amphibious lifestyles in killifishes. Calcification also did not differ between amphibious and fully aquatic species, but terrestrial acclimation caused calcium deposition on the filaments of the killifish Kryptolebias marmoratus , suggesting a possible structural role when out of water. We found strong evidence supporting a role for filament calcification in enhancing aquatic respiratory function. First, acclimation to increased respiratory demands (hypoxia, elevated temperatures) induced calcium deposition on the filaments of K. marmoratus . Next, gentle removal of filament calcification decreased branchial resistance to water flow, indicating disruption of gill basket positioning. Thus, the mechanical properties of the gill filaments appear to play an important and previously unappreciated role in determining fish respiratory function.