Integrin αvβ3 was reported as positive regulators of tumorigenesis and highly expressed in cancer stem cells and kinds of cancers, thus, it is an appealing target for cancer treatment. Nanomedicine with targeting delivery ability has developed rapidly and shown its great therapeutic potential in cancer therapy. Proteins are ideal material for nanomedicine regarding to their excellent biocompatibility, and protein-only self-assembled nanoparticles technology provides a robust method to produce protein nanoparticles. Pro-apoptotic proteins or peptides, such as BAK, have attracted increasing attention in the inhibition of tumor growth. However, the self-assembled nanoparticles of BAK targeting to integrin αvβ3 over-expressed tumor cells need to be investigated. In this study, we designed recombinant proteins with BH3 BAK as active domain and RGD peptides as targeting ligands to self-assemble into protein nanoparticles (named as PN2-1 et al.), then experimentally evaluated the nanoparticle size, fluorescence feature, stability, targeting ability and cytotoxicity to tumor cells in vitro. The results showed that the protein nanoparticles containing RGD peptides had a uniform particle size with an diameter of approximately 23 nm. PN2-1 had notable inhibition to cell proliferation of C6 cells, C26 cells and MCF-7 cells, with a lower IC50 than the nanoparticles which only had BAK motif without RGD peptide. PN2-1 had higher cellular uptake into C6 cells than MCF-7 cells. Our results demonstrate that the RGD peptide could enhance the cytotoxicity of BAK nanoparticles to tumor cells and increase their tumor targeting ability. This study provides an insight into the design and development of integrin αvβ3 targeting protein nanoparticle for cancer treatment.