The successful migration of euryhaline teleost fish from freshwater to seawater requires the up regulation of gill Na⁺,K⁺-ATPase, an enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma ion levels to individuals maintained in freshwater, indicating they successfully acclimated to seawater. This acclimation was associated with an eight-fold increase in gill Na⁺,K⁺-ATPase activity but only a three-fold increase in gill Na⁺,K⁺-ATPase protein number, suggesting other mechanisms may also modulate gill Na⁺,K⁺-ATPase activity. We therefore investigated the influence of membrane composition on Na⁺,K⁺-ATPase activity by examining the phospholipid, fatty acid and cholesterol composition of the gill BLM from freshwater and seawater acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower (~22%) in seawater acclimated char. Gill Na⁺,K⁺-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with % PE and overall % 18:2n6 (linoleic acid) content of the BLM, suggesting gill Na⁺,K⁺-ATPase activity of seawater acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na⁺,K⁺-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured suggesting different lipid protein interactions may exist for char living in each environment.