5,13,19,10 ]eicosane-3,12-dione (MFTZ-1), a new macrolide compound isolated from Streptomyces sp. Is9131, displayed wide cytotoxicity in human tumor cell lines with an average IC 50 of 0.905 Mmol/L. Notably, MFTZ-1 showed significant cytotoxicity in the three multidrug resistance cell lines with an average resistance factor of 2.08. The in vivo experiments showed that MFTZ-1 had inhibitory effects on the human ovarian carcinoma HO-8910 cell line xenotransplanted in nude mice. Further studies showed that MFTZ-1 induced DNA double-strand breaks and triggered mitochondriadependent apoptosis in human leukemia HL-60 cells. Using a yeast genetic system, we found that topoisomerase (Topo) II rather than Topo I was the primary cellular target of MFTZ-1. Most importantly, MFTZ-1 functions as a novel nonintercalative Topo II poison via binding to ATPase of Topo II, characterized by its strong inhibition on the decatenation and relaxation of Topo II. The capacity of MFTZ-1 to stabilize Topo II -DNA covalent complexes was comparable with that of the classic Topo II poison, etoposide. Moreover, using a Topo II catalytic inhibitor aclarubicin and Topo II -deficient HL-60/MX2 cells, we further showed that MFTZ-1 -triggered DNA doublestrand breaks and apoptosis occurred in a Topo IIdependent manner. Together, the well-defined Topo IIpoisoning function and the potent antitumor activity, with the appreciable anti -multidrug resistance action in particular, promises MFTZ-1 as a novel potential Topo II -targeted agent, which merits further research and development.