We have previously reported purification of an extracellular polysaccharide GA3P, D-galactan sulfate associated with L-(+)-lactic acid, produced by a toxic marine microalga Dinoflagellate Gymnodinium sp. A(3) (GA3), and induction thereby of apoptosis on human myeloid leukemia K562 cells. In the present report, we show that the GA3P is a potent inhibitor of DNA topoisomerase (topo) I and topo II, irrespective of the presence or absence of the lactate group. Dextran sulfate also showed similar level of inhibition of topo I and topo II. We also demonstrated that, unlike camptothecin (CPT) or teniposide (VM-26), the inhibition of topo I or topo II by the polysaccharide does not involve accumulation of DNA-topo I/II cleavable complexes, clearly showing that they are not topo poisons but catalytic inhibitors with dual activity. Furthermore, the polysaccharide, when added to the reaction mixture with CPT or VM-26, inhibited stabilization of cleavable complex induced by the latter compounds. In addition, when added to the reaction mixture after the formation of the cleavable complexes by topo poisons, CPT for topo I and VM-26 for topo II, either GA3P or dextran sulfate diminished the amount of the complexes already accumulated, i.e. reversal of the reaction. These results suggest that the polysaccharides bind to the enzymes with high affinities, and that, as for topo I/II inhibition, the GA3P shares a common mechanism with dextran sulfate. As examined in vitro with a human cancer cell line panel, GA3P exhibited significant cytotoxicity against a variety of cancer cells. These findings show that the polysaccharide GA3P would prove to be a potential anticancer chemotherapeutic agent with dual activity of topo I and topo II catalytic inhibition.