The use of natural killer (NK) cells in cell therapy is an attractive next generation strategy for cancer immunotherapy. NK-92 cells (a human NK cell line) have been tested in clinical trial stages, making them an off-the-shelf medicine. Controlling gene expression in NK-92 cells by an artificial delivery system is an available for enhancing NK-92 cell therapy. We report here on the development of a siRNA-loaded lipid nanoparticle (LNP) composed of CL1H6 (CL1H6-LNP), an optimized, pH-sensitive cationic lipid, with efficient gene silencing and low cytotoxicity in NK-92 cells. The hydrophilic head group of the lipid molecule used in preparing these particles largely influences the pKa of the final LNP, and lipids with an amino moiety substituted with a methyl group showed a high gene silencing activity. Compared with myristate and palmitate, the hydrophobic tail of oleate had a high gene silencing activity and cell viability. Analyses of intracellular trafficking indicated that the CL1H6-LNP appeared to escape from the endosomes via membrane fusion, without disrupting the membrane. The mechanism of endosomal escape should contribute to our understanding of efficient gene silencing with a low degree of cytotoxicity. These results therefore suggest that a CL1H6-LNP has promise for delivering siRNA to NK-92 cells.