The purpose of the present study was to develop nanoparticulate drug carrier systems for anticancer drugs and evaluate their efficacy by in vitro anticancer assays and release studies. Herein, diosgenin (DGN) was chemically conjugated to the polymeric carrier via a cleavable ester bond. We developed methoxy poly(ethylene glycol) (MPEG) as the shell, DGN conjugated poly(epsilon-caprolactone) (PCL) as the hydrophobic core as well as amphiphilic block copolymers to design a co-delivery system. The physicochemical properties of the DGN-conjugated PCL-MPEG copolymers were determined by GPC, H-1-NMR, FT-IR, TGA and DSC techniques. Then, Imatinib (ITB) was physically encapsulated by DGN-conjugated PCL-MPEG nanoparticles (NPs) utilizing an emulsion solvent evaporation method. The nanoparticulate formulations were characterized by TEM and DLS. Drug loading and encapsulation efficiency, the in vitro drug release profile, pharmacological evaluation and the stability studies of NPs were investigated. The results showed that the prepared spherical NPs had a uniform particle size (<200 nm), narrow polydispersity index (<0.150), high encapsulation efficacy (60-85%), drug loading capacity (10-15%) and good colloidal stability. The in vitro release profile was influenced by the pH values of the simulated medium and exhibited an initial burst effect followed by a slower and continuous release over a period of 10 days. The in vitro biocompatibility and anticancer activity were evaluated using human fibroblast (L929), leukemia (K-562), osteosarcoma (SAOS-2), and breast carcinoma cells (MCF-7). The cytotoxicity assay showed that the IC50 of ITB-loaded DGN-conjugated NPs were lower compared to the free drugs. Moreover, DGN-conjugated NPs exhibited an obvious synergistic effect by the combination of two anticancer drugs, DGN and ITB. These results suggest that diosgenin-conjugated polymeric NPs may be considered as a promising delivery system for cancer therapy.