To explore the effect of Aclarubicin (ACL) and Mitoxantrone (MTX) combination, we detected the effects of the two drugs on the survival rate of the L929 cell line by cell survival kit and Annexin V/PI double staining. The results showed that ACL reduced MTX induced cell death. In vivo complex of enzyme assay (ICE assay) showed that MTX significantly promoted the formation of TOP2-DNA complexes, but ACL significantly reduced the level of this complex. Western blotting showed that compared with MTX treated cells, the combination of ACL and MTX can significantly reduce the formation of γH2AX protein, indicating that ACL can reduce MTX-induced DNA damage. This suggests that ACL countered MTX induced cell death via reducing TOP2-DNA formation, implicating that ACL might play an antagonistic role against MTX.