Alterations of cellular metabolism play a central role in the development and progression of cancer. Oroxylin A, an active flavonoid of a Chinese traditional medicinal plant, was previously shown to modulate glycolysis in cancer cells. However, the mechanism by which oroxylin A regulates glycolysis is still not well defined. Here, we show that oroxylin A inhibits glycolysis in breast cancer cells via the Sirtuin 3 (SIRT3)-mediated destabilization of hypoxia-inducible factor 1α (HIF1α), which controls glycolytic gene expression. Oroxylin A promotes superoxide dismutase (SOD2) gene expression through SIRT3-regulated DNA-binding activity of FOXO3a and increases the activity of SOD2 by promoting SIRT3-mediated deacetylation. In vivo, oroxylin A inhibits the growth of transplanted human breast tumors associated with glycolytic suppression. These data indicate that oroxylin A inhibits glycolysis-dependent proliferation of breast cancer cells, through the suppression of HIF1α stabilization via SIRT3 activation, providing preclinical information for the cancer therapies of SIRT3 stimulation.