BACKGROUND: Inhibition of the Hedgehog and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathways has been shown to suppress tumor proliferation and stem cell activity. However, the precise role of these pathways in vasculogenic mimicry (VM) of ovarian cancer stem cells (OCSCs) remains unclear. AIMS: To investigate the roles of the PI3K/AKT and Hedgehog signaling pathways in VM formation and the underlying mechanisms in OCSCs. STUDY DESIGN: In vitro and in vivo experimental model. METHODS: OCSCs were induced through serum-free culture of SK-OV-3. Hypoxia-inducible factor-1α (HIF-1α) knockdown was achieved by transfection with sh-HIF-1α. Cells were treated with the PI3K agonist 740 Y-P, the PI3K inhibitor LY294002, the Hedgehog agonist purmorphamine, and the Hedgehog inhibitor cyclopamine under hypoxic conditions. Expression of HIF-1α, epithelial-to-endothelial transition (EET) markers, and components of the PI3K and Hedgehog pathways was analyzed using immunofluorescence and Western blotting. VM capacity was assessed using a Matrigel three-dimensional (3D) culture assay. Cell proliferation and invasion were evaluated by MTS, EdU, and Transwell assays. VM formation was further examined in an OCSC xenograft model. RESULTS: OCSCs accounted for more than 85% of seventh-generation SK-OV-3 cells cultured under serum-free conditions. Hypoxia markedly increased HIF-1α expression, which activated the PI3K and Hedgehog signaling pathways. HIF-1α knockdown suppressed activation of these pathways. Treatment with LY294002 and cyclopamine, as well as HIF-1α knockdown, inhibited hypoxia-induced upregulation of N-cadherin and VE-cadherin, as well as the formation of branching points and 3D channels. Moreover, both LY294002 and cyclopamine significantly reduced cell proliferation, invasion, and VM formation in vitro and in xenografted OCSCs. CONCLUSION: HIF-1α knockdown inhibits activation of the PI3K and Hedgehog signaling pathways, thereby reducing EET and VM formation in hypoxia-induced OCSCs.