Prostate cancer is the most common malignancy in men. For the development of potential anti-prostate cancer agents, a series of arylpiperazine derivatives was synthesized based on previous studies. The target compounds were synthesized via three-step reaction and their in vitro cytotoxic efficacies against a panel of three human prostate cancer cell lines (PC-3, LNCaP, and DU145) and one normal prostate epithelial cell line (WPMY-1) were evaluated by CCK-8 assay. The derivatives 6, 7, 8, 10, 11, 13, 16, 20, 21, 22 and 25 showed important cytotoxic actions against the individual tested cancer cell line with IC50 values <10 μM. The results showed that the o-substituted phenyl group derivatives and m-substituted phenyl group derivatives displayed improved cytotoxic activity against PC-3 cells.