The purpose of this investigation was to evaluate the cytotoxic activity of natural killer (NK) cells isolated from lung cancer patients against the lung cancer (A549) cell line. Two samples of NK1 and NK2 cells (E) were activated, proliferated in vitro, and co-cultured with A549 cells (T) with E:T ratios of 1:1, 2:1, 5:1, 10:1, and 20:1 for 24 and 48 hours, respectively. Results of co-culturing for 24 hours indicated that at the ratios of E: T cells of 1:1, 2:1, and 5:1, respectively, the cytotoxicity of NK cells was still limited, with the survival rate of A549 cells exceeding 90%. However, at a ratio of 10:1 and 20:1, the cytotoxicity of NK cells was obviously demonstrated, with an A549 cell survival rate of 11.54%. With an E:T ratio of 20:1, the A549 cell survival rate dropped to as low as 0.53% after 48 hours of co-cultivation. Consequently, when co-culturing A549 lung cancer cells with natural killer cells, the survival rate of A549 lung cancer cells was significantly affected over time, as was the rate of co- culture.