The discovery of new effective DNA-targeted antitumor agent is needed because of their clinical significance. As acridines can intercalate into DNA and benzimidazoles have the ability to bind in the DNA minor groove, a series of novel benzimidazole acridine derivatives were designed and synthesized to be new DNA-targeted compounds. MTT assay indicated that most of the synthesized compounds displayed good antiproliferative activity, among which compound 8l demonstrated the highest activity against both K562 and HepG-2 cells. Further experiments showed that 8l displayed good DNA-binding capability and inhibited topoisomerase I activity. Moreover, compound 8l could induce apoptosis in K562 cell lines through mitochondrial pathway. These data suggested that compound 8l might be potential as new DNA-binding and apoptosis-inducing antitumor agents.