Quercetin (Que) has consistently been reported to be useful cytotoxic compound in vivo and in vitro, but little is known on its metabolites. Here we examined and compared cytotoxic effect of Que and its water-soluble metabolites, isorhamnetin (IS) and isorhamnetin-3-glucuronide (I3G) in human breast cancer MCF-7 cells, and explain their tumor-inhibitory mechanism and structure-function relationship. The results showed that Que, IS and I3G could dose-dependently inhibit the growth of MCF-7 cells, and the cytotoxic effect was ranked as Que > IS > I3G. Furthermore, Que, IS and I3G mediated the cell-cycle arrest principally in S phase, followed by the decrease in the number of G0/G1 and G2/M, and 70.8%, 68.9% and 49.8% MCF-7 tumor cells entered early phase apotosis when treated with 100 µM Que, IS and I3G for 48 h, respectively. Moreover, induction of apoptosis by Que, IS and I3G were accompanied with the marginal generation of intracellular ROS. Given these results, Que, IS and I3G possess strong cytotoxic effect through a ROS-dependent apoptosis pathway in MCF-7 cells.