Tumor cells in hypoxic conditions control cancer metabolism and angiogenesis by expressing HIF-1α. Tanshinone is a traditional Chinese medicine that has been shown to possess antitumor properties and exerts a therapeutic impact on angiogenesis. However, the precise molecular mechanism responsible for the antitumor activity of 3-Hydroxytanshinone (3-HT), a type of tanshinone, has not been fully understood. Therefore, our study aimed to investigate the mechanism by which 3-HT regulates the expression of HIF-1α. Our findings demonstrate that 3-HT inhibits HIF-1α activity and expression under hypoxic conditions. Additionally, 3-HT inhibits hypoxia-induced angiogenesis by suppressing the expression of VEGF. Moreover, 3-HT was found to directly bind to α-enolase, an enzyme associated with glycolysis, resulting in the suppression of its activity. This inhibition of α-enolase activity by 3-HT leads to the blockade of the glycolytic pathway and a decrease in glycolysis products, ultimately altering HIF1-α expression. Furthermore, 3-HT negatively regulates the expression of HIF-1α by altering the phosphorylation of AMP-activated protein kinase (AMPK). Our study's findings elucidate the mechanism by which 3-HT regulates HIF-1α through the inhibition of the glycolytic enzyme α-enolase and the phosphorylation of AMPK. These results suggest that 3-HT holds promise as a potential therapeutic agent for hypoxia-related angiogenesis and tumorigenesis.