Aurora A kinase, a member of the Aurora kinase family, is frequently overexpressed in various human cancers. In addition, Overexpression of Aurora A kinase is associated with drug resistance and poor prognosis in many cancers including breast cancer. Therefore, Aurora A kinase has been considered as an attractive anticancer target for the treatment of human cancers. Herein, A series of 6-(2-amino-1H-benzo[d]imidazole-6-yl)quinazolin-4(3H)-one derivatives were designed, synthesized, and evaluated as Aurora A kinase inhibitors. The cell-based cytotoxicity assays showed that compound 16h was the most potent cytotoxic agent against all tested cancer cells and had a lower IC value than ENMD-2076 against MDA-MB-231 cells. Meanwhile, Aurora A kinase assay and Western blot analysis showed that 16h inhibited Aurora A kinase with an IC value of 21.94 nM and suppressed the phosphorylation of Histone H3 on Ser10 and Aurora A kinase on Thr288, which were consistent with the activation of Aurora A kinase. Accordingly, 16h caused aberrant mitotic phenotypes and obvious G2/M phase arrest in MDA-MB-231 cells and induced caspase-dependent apoptosis in MDA-MB-231 cells. These results demonstrated that 16h is a potential candidate for the development of anticancer agents targeting Aurora A kinase.