Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer death in men. Regarding that prostate cancer is the most common form of cancer in men and is the second leading cause of cancer mortality, paclitaxel, as a chemotherapeutic agent with a wide spectrum of antitumor activity, could be utilized in treatment of this malignancy. Paclitaxel side effects are severe hypersensitivity reactions, neurotoxicity, and nephrotoxicity. Today’s decline of side effects and increase in efficacy of chemotherapeutic agents by applying nanotechnology in medicine is the target of scientists. Niosomes or nonionic surfactant vesicles are nano vehicles utilized in drug delivery systems. Niosomes are prepared by various methods. Our present work investigated the efficiency of encapsulation of paclitaxel in noisome (Nio-PTX) as a novel vesicular drug delivery system and cytotoxic effects on PC-3 prostate cancer cell line. In this study, paclitaxel loaded niosome was prepared by thin film hydration method. The characterization tests that included dynamic light scattering (DLS) and UV-Vis spectrophotometry were employed to evaluate the quality of the nanocarriers. Percent of encapsulation paclitaxel prepared with sorbitane monostearate and cholesterol was 99.4%. In addition, the polydispersity index, mean size diameter and zeta potentials of niosomal paclitaxel nanoparticles were found to be 0.203 ± 0.012, 119.7 ± 2.5 nm and -4 ± 0.34, respectively. Cytotoxicity of niosomal paclitaxel nanoparticles and free paclitaxel on human prostate cancer cell line PC-3 after 24 hours were studied by MTT assay to determine cell viability. The results demonstrated that a 1.5∼fold reduction in paclitaxel concentration was measured when the paclitaxel administered in nanoniosome compared to free paclitaxel solution in PC3 human prostate cancer cell line. As a result, the nanoparticle-based formulation of paclitaxel has high potential as an adjuvant therapy for clinical usage in human prostate cancer therapy