DNA topoisomerase I (Top1) is an essential nuclear enzyme and a validated target for anticancer agent screening. In a previous study, we found that indolizinoquinoline-5,12-dione derivatives show significant biological activity against several human cancer cell lines. To understand their mechanism of inhibition of cancer cell growth, one indolizinoquinoline-5,12-dione derivative, CY13II, was further studied as lead. Our present results indicate that CY13II shows more potent antiproliferative activity against K562 cells than camptothecin. Additionally, K562 cells were arrested in G2/M, and their growth rate decreased after treatment with CY13II at micromolar concentration. Biochemical Top1 assays indicate that CY13II exhibits a different inhibitory mechanism from camptothecin. Unlike camptothecin, CY13II specifically inhibits the catalytic cleavage activity of Top1 instead of forming the drug-enzyme-DNA covalent ternary complex.