FSH stimulation of granulosa cells (GCs) results in increased hypoxia-inducible factor (HIF)-1 protein levels and HIF-1 activity that is necessary for up-regulation of certain FSH target genes including vascular endothelial growth factor (VEGF). We report that the role of the phosphatidylinositol (PI)-3-kinase/AKT pathway in increasing HIF-1 protein in FSH-stimulated GCs extends beyond an increase in mammalian target of rapamycin (mTOR)-stimulated translation. FSH increases phosphorylation of the AKT target mouse double minute 2 (MDM2); a phosphomimetic mutation of MDM2 is sufficient to induce HIF-1 activity. The PI3-kinase/AKT target forkhead box-containing protein O subfamily 1 (FOXO1) also effects the accumulation of HIF-1 as evidenced by the ability of a constitutively active FOXO1 mutant to inhibit the induction by FSH of HIF-1 protein and HIF-1 activity. Activation of the PI3-kinase/AKT pathway in GCs by IGF-1 is sufficient to induce HIF-1 protein but surprisingly not HIF-1 activity. HIF-1 activity also appears to require a PD98059-sensitive protein (kinase) activity stimulated by FSH that is both distinct from mitogen-activated extracellular signal-regulated kinase (ERK) kinase1/2 or 5 and independent of the PI3-kinase/AKT pathway. These results indicate that FSH-stimulated HIF-1 activation leading to up-regulation of targets such as VEGF requires not only PI3-kinase/AKT-mediated activation of mTOR as well as phosphorylation of FOXO1 and possibly MDM2 but also a protein (kinase) activity that is inhibited by the classic ERK kinase inhibitor PD98059 but is not ERK1/2 or 5. Thus, regulation of HIF-1 activity in GCs by FSH under normoxic conditions is complex and requires input from multiple signaling pathways.