Survival of prolonged anoxia requires a balance between cellular ATP demand and anaerobic ATP supply from glycolysis, especially in critical tissues such as the brain. To add insight into the ATP demand of the brain of the anoxia-tolerant red-eared slider turtle (Trachemys scripta) during prolonged periods of anoxic submergence, we quantified and compared the number of Na-K-ATPase units and their molecular activity in brain tissue from turtles acclimated to either 21°C or 5°C and exposed to either normoxia or anoxia (6h 21°C; 14days at 5°C). Na-K-ATPase activity and density per g tissue were similar at 21°C and 5°C in normoxic turtles. Likewise, anoxia exposure at 21°C did not induce any change in Na-K-ATPase activity or density. In contrast, prolonged anoxia at 5°C significantly reduced Na-K-ATPase activity by 55%, which was largely driven by a 50% reduction of the number of Na-K-ATPase units without a change in the activity of existing Na-K-ATPase pumps or α-subunit composition. These findings are consistent with the "channel arrest" hypothesis to reduce turtle brain Na-K-ATPase activity during prolonged, but not short-term anoxia, a change that likely helps them overwinter under low temperature, anoxic conditions.