Natural killer (NK) cells provide a powerful weapon mediating immune defense against viral infections, tumor growth, and metastatic spread. NK cells demonstrate great potential for cancer immunotherapy; they can rapidly and directly kill cancer cells in the absence of MHC-dependent antigen presentation and can initiate a robust immune response in the tumor microenvironment (TME). Nevertheless, current NK cell-based immunotherapies have several drawbacks, such as the requirement for ex vivo expansion of modified NK cells, and low transduction efficiency. Furthermore, to date, no clinical trial has demonstrated a significant benefit for NK-based therapies in patients with advanced solid tumors, mainly due to the suppressive TME. To overcome current obstacles in NK cell-based immunotherapies, we describe here a non-viral lipid nanoparticle-based delivery system that encapsulates small interfering RNAs (siRNAs) to gene silence the key intrinsic inhibitory NK cell molecules, SHP-1, Cbl-b, and c-Cbl. The nanoparticles (NPs) target NK cells in vivo, silence inhibitory checkpoint signaling molecules, and unleash NK cell activity to eliminate tumors. Thus, the novel NP-based system developed here may serve as a powerful tool for future NK cell-based therapeutic approaches.