In this study, two series of novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives containing an α,β-unsaturated lactone fragment were synthesized and screened for Topo I inhibition and antitumor activity. The topoisomerase I inhibitory activities and cytotoxicities against three human cancer cell lines (MCF-7,Hela,A549) were evaluated. The results revealed that series 2, compounds bearing an exocyclic double bond on the furanone ring, generally showed more potent activity than series 1, compounds lacking an exocyclic double bond. Several compounds of series 2 possess significant Topo I inhibitory activity and potent antiproliferative activity against cancer cell lines. Further mechanism studies of the most active compound of series 2 (B-15) indicated that synthetic compounds can not only stabilize the drug-enzyme-DNA covalent ternary complex as well as camptothecin, but also interfere with the binding between Topo I and DNA. The binding patterns of these compounds with Topo I and structure-activity relationships are discussed.