Fludarabine is the major drug against chronic lymphoblastic leukemia. However, the effects of fludarabine on solid tumors remain unclear. We reported previously that fludarabine inhibited expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human ovarian cancer cells. The present study aims to investigate, (1) the mechanisms that fludarabine inhibits expression of HIF-1alpha and (2) the effects of fludarabine on tumor cells survival under hypoxia. Human hepatoma HepG2 cells were tested. We found that fludarabine reduced HIF-1alpha stability through prolyl hydroxylation. Fludarabine suppressed hypoxia-induced expression of VEGF and its receptor KDR. It inhibited VEGF-induced activation of AKT and ERK signaling and reduced hypoxia-enhanced HepG2 cell survivability. HepG2 cells were much more sensitive to fludarabine treatment under hypoxia than under nomoxia. These results suggest that fludarabine reduces survivability of HepG2 cells via VEGF signaling under hypoxia.