We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 ± 0.19 mg O2/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD+]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADPfree], redox state, or [pyruvate]). Hypoxia exposure caused a ∼65% decrease in gill Na+/K+ ATPase activity, which was not matched by changes in Na+/K+ ATPase α-subunit protein abundance indicating post-translational modification of Na+/K+ ATPase was responsible for the decrease in activity. Despite decreases in gill Na+/K+ ATPase activity, plasma [Na+] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na+/K+ ATPase α-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na+/K+ ATPase α-subunit relative to other genes.