Following the Deepwater Horizon oil spill, approximately 7 million liters of the dispersant Corexit 9500A were released to promote oil biodegradation by breaking up surface oil slick formation. This process is accomplished via amphipathic anionic surfactants within dispersants that facilitate the mixing of aqueous and lipid phases. However, the amphipathicity of Corexit may also cause it to interact with biological membranes like the gill, impairing gill function and ultimately disrupting physiological processes mediated by it, such as osmoregulation. The goal of this study was to investigate the osmoregulatory effects and toxicity of Corexit in Gulf killifish. Killifish at the embryonic, larval, juvenile, and adult life stages were exposed to Corexit in water of different salinities to assess the interactive effects of ontogeny and salinity on Corexit toxicity. Corexit was not toxic to embryos except when exposed in hyperosmotic water where it had negligible effects; however, its toxicity to killifish increased dramatically following hatch, showing its greatest deleterious effects in adults. Corexit tended to increase sodium and chloride burdens in killifish when exposed in hyperosmotic waters and reduced whole-body and plasma ion concentrations in fish exposed to hypoosmotic waters. However, Corexit exposure at hyperosmotic salinities resulted in an increased differential accumulation of sodium over chloride as killifish matured. These findings suggest that Corexit may impair gill structure or alter specific components of osmoregulatory function, thus impacting osmoregulation in hypersosmotic and hypoosmotic waters, potentially impairing survival during osmotic challenges. Furthermore, the magnitude of these impacts continues to increase concomitant with gill ontogeny.