3-Chloromethylene-6-fluorothiochroman-4-one (CMFT) is a novel thiochromanones derivative that has better anti-tumor activity. In this paper, we will compare the antitumor activity of the cis-trans isomers, and explore their inhibiting effects on human topoisomerase I and topoisomerase II in cell free reaction system. The MTT method was used to study inhibition rates; the AO/EB double staining and TUNEL assay was used to assess proportion of apoptotic cells. The inhibition of CMFT to Topo I and II could be identified by adding CMFT solutions to Topo-DNA reaction mixtures and observing the relative quantities of relaxed strands and supercoils in electrophoresis assay. Results showed that CMFT had dramatic anti-tumor activities at low concentrations and the activity of CMFT trans-isomer is more significant. Use of AO/EB double staining and TUNEL indicated that CMFT induces apoptosis. DNA relaxation assays and DNA cleavage and relegation assays were performed and showed a higher potential to interact with topoisomerase I (Topo I) and topoisomerase II (Topo II) and it was verified that CMFT is a Topo poison which could be one of the mechanisms that induce cell apoptosis. Our results provide preliminary data for further investigation for the mechanism of CMFT of the apoptotic mechanism.