Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by limited therapeutic options and a pronounced tendency for relapse. PX-478, a novel inhibitor of hypoxia-inducible factor 1-alpha (HIF-1α), has demonstrated antitumor activity across various cancer models, but its specific role in AML remains unexplored. This study aimed to explore the potential target and mechanism of PX-478-induced AML cell apoptosis. First, PX-478 induced AML cell apoptosis in vitro under hypoxia via modulation of the Bcl-2 family and activation of the mitochondria-mediated caspase cascade, exhibiting a concentration-dependent effect. Additionally, in vivo administration of PX-478 led to notable inhibition of subcutaneous AML xenograft growth in mice, coupled with increased tumor cell apoptosis. RNA sequencing and cellular studies revealed downregulation of the PI3K/AKT/mTOR signaling pathway in PX-478-treated cells. Consistently, cellular studies also implicated PI3K/AKT/mTOR pathway in PX-478-induced AML cell apoptosis. Furthermore, by screening for RNA sequencing differential genes and subsequent experimental verification, Glycogen branching enzyme 1 (GBE1) may be involved in PX-478-induced apoptosis in AML cells. We found that inhibiting GBE1 expression in AML cells (siGBE1) led to downregulation of the PI3K/AKT/mTOR pathway and induced apoptosis. In experiments using AML cells with reduced GBE1 expression (shGBE1), PX-478 treatment did not further downregulate the pathway or enhance apoptosis. Re-expression of GBE1 in shGBE1 cells alleviated apoptosis and reduced PX-478- induced apoptosis and pathway downregulation. In conclusion, our findings provide convincing evidence that PX-478 induces apoptosis by inhibiting the PI3K/AKT/mTOR pathway through downregulation of GBE1 in AML cells.