There are seven extant members of the Order Lepisosteiformes commonly known as gar distributed in North and Central America. This group of fishes is cultured as a food-fish and for conservation stocking. Members of this family possess many primitive characteristics and span a wide range of habitats, including freshwater rivers, estuaries, and saline coastal marshes. Euryhalinity is important to many populations, but relatively little is known about adaptations to compensate for salinity changes, information important for their culture and introduction to natural habitats. Therefore, several experiments were conducted to better understand physiological changes associated with acclimation to a range of salinity in alligator gar (Atractosteus spatula). To guide an understanding of changes in mucosal composition which may be important for general health and immune defenses, mucus peptides were compared between gar acclimated to freshwater (0 ppt) or saline water (20 ppt). Scanning and transmission electron microscopy were used to examine changes in gill ultrastructure. Capabilities to respond to a handling stressor were evaluated using blood measurements of primary and secondary stress responses. In addition, to understand the ability to rapidly compensate for a change in salinity, gill and kidney proteomic responses were evaluated during a time-course of salinity acclimation, alongside blood osmoregulatory measurements. Collectively, results of this study will be discussed in the context of providing a baseline of understanding important for guiding culture practices for gar.