A series of oxoisoaporphine derivatives with topoisomerase I inhibition and cytotoxic activities. Among them, compound 14 showed the most potent cytotoxic activity against all cancer cell lines tested, and substantially lower cytotoxicity to LO2 cells. Molecular docking studies, dynamics simulation and a follow-up enzyme inhibition assay indicated that 14 could interfere with DNA and significantly inhibit the activity of topoisomerase I. Further mechanistic studies revealed that 14 could arrest cell cycle at the G1 phase, and finally killed MCF-7 cells via apoptosis. In addition, 14 exhibited remarkable chemoreversal ability on multidrug-resistant MCF-7/ADR breast cancer cells. Some of its mechanisms may be related to inhibition of MCF-7/ADR P-gp-mediated Rhodamine (Rh123) efflux function and expression level, as well as inhibition of ROS, increase of ADR accumulation in MCF7/ADR cells, and enhancement of ADR in inducing apoptosis of MCF7/ADR cells. As 14 has little toxic and side effects, it may have the potential for further research.