Objective: To compare the difference in proliferation and cytotoxicity of A-NK cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assist effect of IL-12 on A-NK/IL-2 treatment and the morphology character of NK cells. Methods: Cellular proliferation was compared by MTT method in vitro. The morphology of the target cells killed by adherent natural killer cells (A-NK) was observed through electroscope. Result: All of the A-NK cells cultured in different conditions could rapidly proliferate (p>0.05) and kill target cells (p>0.05), death patterns of tumor target cells were necrosis and apoptosis. Conclusion: These data indicate that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells in vitro, A-NK cells activated by both IL-2 and IL-12 was superiority to the IL-2 activated cells, and could reduce side effects by high dose IL-2. The studies provided a new experimental basis for experimental and clinical treatment of A-NK cell in future.