Background: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) induces the expression of glycolysis-related genes, which plays a direct and key role in Warburg effect. In a recent study, honokiol (HNK) was identified as one of the potential agents that inhibited the HIF-1 alpha signaling pathway. Because the HIF- 1 alpha pathway is closely associated with glycolysis, we investigated whether HNK inhibited HIF-1 alpha-mediated glycolysis. Methods: The effects of HNK on HIF-1 alpha-mediated glycolysis and other glycolysis-related genes' expressions, cancer cells apoptosis and tumor growth were studied in various human breast cancer models in vitro and in vivo. We performed the following tests: extracellular acidification and oxygen consumption rate assays, glucose uptake, lactate, and ATP assays for testing glycolysis; WST-1 assay for investigating cell viability; colony formation assay for determining clonogenicity; flow cytometry for assessing cell apoptosis; qPCR and Western blot for determining the expression of HIF-1 alpha, GLUT1, HK2 and PDK1. The mechanisms of which HNK functions as a direct inhibitor of HIF-1 alpha were verified through the ubiquitination assay, the Co-IP assay, and the cycloheximide (CHX) pulse-chase assay. Results: HNK increased the oxygen consumption rate while decreased the extracellular acidification rate in breast cancer cells; it further reduced glucose uptake, lactic acid production and ATP production in cancer cells. The inhibitory effect of HNK on glycolysis is HIF-1 alpha-dependent. HNK also downregulated the expression of HIF-1 alpha and its downstream regulators, including GLUT1, HK2 and PDK1. A mechanistic study demonstrated that HNK enhanced the self-ubiquitination of HIF-1 alpha by recruiting two E3 ubiquitin ligases (UFL1 and BRE1B). In vitro, HNK inhibited cell proliferation and clonogenicity, as well as induced apoptosis of cancer cells. These effects were also HIF1 alpha-dependent. In vivo, HNK inhibited tumor growth and HIF-1 alpha-mediated glycolysis. Conclusion: HNK has an inhibitory effect on HIF-1 alpha-mediated glycolysis in human breast cancer. Our research revealed a new mechanism of HNK as an anti-cancer drug, thus representing a novel strategy to improve the prognosis of cancer.