BACKGROUND: Chitosan can be modified to increase the efficiency of the delivery of chemical drugs, nucleic acids, or proteins. Sodium tripolyphosphate (TPP) is a noncytotoxic and polyanionic crosslinker that binds with the positively charged ions of chitosan. DsNKG2D-IL-15 is a fusion protein that exerts promising antitumor effects via lymphocyte activation. The extracellular domains of double NKG2D is linked to IL-15. METHODS: To increase the stability and efficiency of dsNKG2D-IL-15 protein, the fusion protein was encapsulated in nanoparticles based on chitosan pre-modified with N-(2-hydroxy) propyl-3-trimethyl ammonium (HTCC). Moreover, the biological activity of protein nanoparticle was evaluated on the mouse lymphocyte and mouse tumor model . RESULTS: TPP sharply promoted the HTCC chitosan encapsulating efficiency (85-95%) with dsNKG2D-IL-15. The protein nanoparticle displayed a spherical shape with a diameter of 200-400 nm and zeta-potential value of 15.6±4.82 mV. DsNKG2D-IL-15 could be released from the nanogel within 72 h. In addition, the protein biological activity for lymphocyte activation was maintained. Natural killer (NK) and CD8 T cells increased the activity of IFN-γ production and degranulation after incubation with the dsNKG2D-IL-15-HTCC-TPP nanoparticle . Treatment with dsNKG2D-IL-15 nanoparticles exhibited better effects of inhibiting tumor growth and prolonging the life span of B16BL6-MICA tumor-bearing mice than by using the dsNKG2D-IL-15 protein alone. CONCLUSIONS: The dsNKG2D-IL-15 protein nanoparticle exhibited notable effects of lymphocyte activation and tumor inhibition. The protein nanoparticle could be developed further for tumor therapy in clinical practice.