The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase-domain containing enzymes (PHD), whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. Addition of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential(_mito) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, _mito is partially maintained during metabolic inhibition and recovers completely upon washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse _mito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of _mito in PHI-treated myocytes, while a role for the 'reverse mode' operation of the F₁Fo-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only ~15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of pre-insult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult.