Over recent years, metal oxide nanoparticles have been extensively investigated due to their potential biomedical applications. This study aimed to describe a simple and eco-friendly method for the green synthesis of cerium oxide nanoparticles (CeO2 NPs) using Falcaria vulgaris (F. vulgaris) leaf extract as a capping agent and to evaluate their anti-cancer effects on human prostate cancer cells (PC3). The phyto-synthesized CeO2 NPs were characterized by UV–visible spectroscopy, X-ray diffraction, transmission electron microscope, Fourier transform infrared spectroscopy, scanning electron microscope, and energy-dispersive X-ray spectroscopy. The UV-Visible spectrum showed a peak at 313 nm with a band gap energy of 3 eV. Microscopic results revealed that CeO2 NPs mainly were spherical. In addition, the XRD pattern confirmed the crystalline structure of green synthesized CeO2 NPs with an average crystalline size of around 19.5 nm. In biological experiments, the results of the resazurin assay indicated the significant cytotoxic activity of CeO2 NPs against PC3 cells with an IC50 at 113.6 μg/ mL. The real-time PCR results revealed that CeO2 NPs effectively regulate PC3 cell proliferation, apoptosis, and migration by modifying the expression of cell cycle-related proteins (c-Myc, cyclin D1, p21), and apoptosis-related genes (Bcl-2 and Bax), and migration-related genes (MMP-2 and MMP-9). Overall, these findings suggest that the green fabricated CeO2 NPs from F. Vulgaris can be used as a novel therapeutic agent for prostate cancer treatment.