Diosgenin (DGN) is a natural bioactive steroidal saponin from therapeutic herbs and it exhibits potential anticancer efficacy. Here, we report the preparation of poly lactide-co-glycolide (PLGA)-encapsulated diosgenin nanoparticles (PLGA-DGN NPs) and characterization of their physicochemical nature, and morphology as well as inspect their drug-loading capability, in vitro drug release, pharmacokinetics, bio-distribution, safety profile. The in vitro and in vivo antineoplastic activities of PLGA-DGN NPs were also examined against the MCF-7 breast cancer cell line and in the Ehrlich ascites carcinoma (EAC) tumor xenograft model, respectively. The results displayed an appropriate size (∼147 nm), spherical shape, and a narrow polydispersity index. The nanoparticles showed a high encapsulation efficacy (78%), loading capacity (8%), good colloidal stability, and an initial burst release at an acidic medium. The nanoparticles exhibited significant cytotoxic activity at their IC50 dose compared to diosgenin. PLGA-DGN NPs facilitated cellular internalization of DGN in human breast cancer MCF-7 cells and extended the blood circulation time showing an excellent pharmacokinetic profile. Bio-distribution study revealed that PLGA-DGN NPs could significantly enhance DGN accumulation in tumor tissue. The safety profile of the PLGA-DGN NPs was confirmed in histopathologic, hematologic, and immunologic parameters of Swiss albino mice. At the same time, an effective antiangiogenic, and antiproliferative potential of PLGA-DGN NPs were exhibited in mice tumor xenograft model by showing tumor regression and down-regulating CD31 and Ki-67 expression. Therefore, PLGA-DGN NPs could be utilized as a promising anticancer drug, opening up a new avenue for further research.