Aurora-B kinase overexpression plays important roles in the malignant progression of prostate cancer (PCa). AZD1152-HQPA, as an inhibitor of Aurora-B, has recently emerged as a promising agent for cancer treatment. In this study, we aimed to investigate the effects of AZD1152-HQPA on reactive oxygen species (ROS) generation and mitochondrial function in PCa. We used AZD1152-HQPA (Barasertib), a highly potent and selective inhibitor of Aurora-B kinase. The effects of AZD1152-HQPA on cell viability, DNA content, cell morphology, and ROS production were studied in the androgen-independent PC-3 PCa cell line. Moreover, the mitochondrial copy number and the expression of genes involved in cell survival and cancer stem cell maintenance were investigated. We found that AZD1152-HQPA treatment induced defective cell survival, polyploidy, micronuclei formation, cell enlargement, and cell death by significant overexpression of p73, p21 and downregulation of cell cycle-regulatory genes in a drug concentration-dependent manner. Moreover, AZD1152 treatment led to an excessive ROS generation and an increase in the mitochondrial copy number not only in PC-3 but also in several other malignant cells. AZD1152 treatment also led to downregulation of genes involved in the maintenance of cancer stem cells. Our results showed a functional relationship between the aurora kinase inhibition, an increase in mitochondrial copy number, and ROS generation in therapeutic modalities of cancer. This study suggests that the excessive ROS generation may be a novel mechanism of cytotoxicity induced by the aurora kinase inhibitor, AZD1152-HQPA.