Nuclear respiratory factor-1 (NRF-1) is integral to the transcriptional regulation of mitochondrial biogenesis but its control over various respiratory genes overlaps other regulatory elements including those involved in O² sensing. Aerobic metabolism generally suppresses hypoxia-sensitive genes, e.g. via hypoxia-inducible factor-1 (HIF-1), but mutations in Complex II—succinate dehydrogenase (SDH), a tumor suppressor, stabilize HIF-1, producing pseudo-hypoxia. In aerobic cardiomyocytes, which rely on oxidative phosphorylation, we tested the hypothesis that NRF-1 regulates Complex II expression and opposes hypoxia-inducible factor-1. NRF-1 gene silencing blocked aerobic succinate oxidation, increasing nuclear HIF-1 protein prior to loss of Complex I function. We postulated that NRF-1 suppression either specifically decreases the expression of one or more SDH subunits and increases succinate availability to regulate HIF-1 prolyl hydroxylases, or stimulates mitochondrial reactive oxygen production, which interferes with HIF-1 degradation. Using promoter analysis, gene silencing, and chromatin immunoprecipitation, NRF-1 was found to bind to the gene promoters of two of four nuclear-encoded Complex II subunits: SDHa and SDHd, but the enzyme activity was dynamically regulated through the catalytic SDHa flavoprotein. Complex II was inactivated by SDHa silencing, which led to aerobic HIF-1 stabilization, nuclear translocation, and enhanced expression of glucose transporters and heme oxygenase-1. This was unrelated to mitochondrial ROS production, reversible by high -ketoglutarate concentrations, and coherent with regulation of HIF-1 by succinate reported in tumor cells. These findings disclose a novel role for NRF-1 in transcriptional control of Complex II and prevention of pseudo-hypoxic gene expression in aerobic cardiac cells.