Aurora kinase A has been demonstrated to be involved in the malignant progression of many types of cancer including prostate cancer, we therefore hypothesized that Aurora kinase A might work as a valuable target for prostate cancer treatment. To test this hypothesis, we used DNAzyme technology to inhibit Aurora kinase A expression and evaluated the effects of DNAzymes as therapeutic agents to treat prostate cancer. In an in vitro cleavage assay, we found that a DNAzyme (DZ2) targeting Aurora kinase A could effectively cleave Aurora kinase A mRNA. When transfected into the prostate cancer cell line PC3, DZ2 was found to strongly inhibit the expression of Aurora kinase A examined by western blot analysis, and thus suppressed cell growth, arrested the progression of cell cycle, induced cell apoptosis and attenuated cell migration, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, flow cytometry and Boyden chamber assay. Through in vivo study, we also found that DZ2 could significantly inhibit the growth of human prostate cancer xenografts in nude mice. In conclusion, DZ2 could effectively attenuate malignant progression of prostate cancer both in vitro and in vivo, suggesting that DNAzyme targeting Aurora kinase A may be used as a valuable therapy to treat prostate cancer.