Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7-trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti-tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein-topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO2 for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein-topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post-treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment-induced apoptosis using caspase-3 and -9 binding assays and (iii) treatment-induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein-topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase-3 and -9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein-topotecan combination treatment. Treatments involving genistein-topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.