Hypoxia initiates an intracellular signaling pathway leading to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activity is regulated through different mechanisms involving stabilization of HIF-1α, phosphorylations, modifications of redox conditions, and interactions with coactivators. However, it appears that some of these steps can be cell type-specific. Among them, the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the regulation of HIF-1 by hypoxia remains controversial. Here, we investigated the activation state of PI3K/Akt/glycogen synthase kinase 3β (GSK3β) in HepG2 cells. Increasing incubation times in hypoxia dramatically decreased both the phosphorylation of Akt and the inhibiting phosphorylation of GSK3β. The PI3K/Akt pathway was necessary for HIF-1α stabilization early during hypoxia. Indeed, its inhibition was sufficient to decrease HIF-1α protein level after 5-h incubation in hypoxia. However, longer exposure (16 h) in hypoxia resulted in a decreased HIF-1α protein level compared with early exposure (5 h). At that time, Akt was no longer present or active, which resulted in a decrease in the inhibiting phosphorylation of GSK3β on Ser-9 and hence in an increased GSK3β activity. GSK3 inhibition reverted the effect of prolonged hypoxia on HIF-1α protein level; more stabilized HIF-1α was observed as well as increased HIF-1 transcriptional activity. Thus, a prolonged hypoxia activates GSK3β, which results in decreased HIF-1α accumulation. In conclusion, hypoxia induced a biphasic effect on HIF-1α stabilization with accumulation in early hypoxia, which depends on an active PI3K/Akt pathway and an inactive GSK3β, whereas prolonged hypoxia results in the inactivation of Akt and activation of GSK3β, which then down-regulates the HIF-1 activity through down-regulation of HIF-1α accumulation.