Quercetin (Qu) is known as a dietary antioxidant with numerous bioactivities, but its function in anti-cancer has not been fully investigated. Here, we show that Qu at low doses (≤10 μM) significantly enhances the inhibition of 10-hydroxy camptothecin (HCPT) on the proliferation of MCF7, BGC823 and HepG2 cells. A plasmid DNA relaxation assay indicates that the inhibition of HCPT on the catalytic activity of topoisomerase I (Topo I) is increased by Qu at 10 μM. Compared to the treatment by Qu or HCPT alone, phosphorylation at Ser139 of γH2A.X in MCF7 cells starts to increase significantly (P < 0.05) at 6 h when treated by the combination of 10 μM Qu and 0.62 μM HCPT. Moreover, the combinational group successively arrests MCF7 cells at G1, S and G2/M phases from 12 h to 48 h via up-regulation of p21 and induces apoptosis at 24 h by triggering intrinsic cell death pathways. In addition, the inhibition effects of the combinational group on the proliferation of MCF7 cells are eliminated by pretreatment with 100 μM Z-VAD-FMK (a caspase inhibitor). Finally, by using nude mice xenografting assay of MCF7 cells, we demonstrate that tumor inhibition rates of combinational group are significantly higher than single-drug group. In summary, the synergic anti-cancer mechanism of Qu and HCPT in MCF7 cells is through the combined inhibitory effects of Qu and HCPT on Topo I, which synergistically induce cell cycle arrest and apoptosis by triggering DNA damage.