Prostaglandin E₂ (PGE₂) has been implicated as an inducer of angiogenesis in human colon cancer. Here, we demonstrate that PGE₂ exposure induces the expression of vascular endothelial growth factor (VEGF) mRNA in HCT116 human colon carcinoma cells that is mediated by the transcriptional activator hypoxia-inducible factor 1 (HIF-1). PGE₂ exposure induces the phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Pharmacologic inhibition of ERK phosphorylation blocks the induction of VEGF mRNA and HIF-1α protein expression in response to PGE₂ stimulation. Inhibition of C-SRC tyrosine kinase activity also blocks PGE₂-induced HIF-1α protein and VEGF mRNA expression without blocking ERK phosphorylation. In contrast, phosphorylation of AKT is dependent on ERK and C-SRC activity. Thus, the activity of multiple signal transduction pathways is required for the HIF-1-mediated induction of VEGF expression in colon cancer cells exposed to PGE₂.