This study is intended to characterize the cellular target of gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hurburyi tree, which possesses potent in vitro and in vivo antitumor activities. The antiproliferative activity of GA was further confirmed here in a panel of human tumor cells and multidrug-resistant cells. We found that GA significantly inhibited the catalytic activity of topoisomerase (Topo) II and, to a comparatively less extent, of Topo I, without trapping and stabilizing covalent topoisomerase-DNA cleavage complexes. Down-regulation of Topo IIalpha but not Topo I and Topo IIbeta, reduced GA-induced apoptosis and the phosphorylation of c-Jun, and restored cell proliferation upon GA treatment. Moreover, GA antagonized etoposide-induced DNA damage and abrogated the antiproliferative activity of etoposide, whereas it did not affect camptothecin-induced DNA damage. By dissecting the actions of GA on the individual steps of Topo IIalpha catalytic cycle, we found that GA inhibited DNA cleavage and ATP hydrolysis. Moreover, GA directly bound to the ATPase domain of Topo IIalpha, and may share common binding sites with ATP. The results reported here show that GA exerts its antiproliferative effect by inhibiting the catalytic activity Topo IIalpha. They also indicate that GA inhibits Topo IIalpha-mediated DNA cleavage and modulate the activity of Topo II poisons, which provide rationale for further clinical evaluation of GA.