Disclosed is a method for producing a large amount of natural killer cells and the use of the natural killer cells as an anticancer agent. The method produces fresh NK cells with high purity within a short time, and can also produce cold-preserved NK cells and thawed cryopreserved NK cells having efficacy comparable to the fresh NK cells. NK cells having efficacy comparable to the fresh NK cells can also be produced from cryopreserved CD3-negative cells. The fresh NK cells, cold-preserved NK cells and cryopreserved NK cells exhibit therapeutic effects against various cancers, including colorectal cancer, lung cancer, liver cancer, pancreatic cancer and leukemia, indicating these NK cells are effective as cellular therapeutic agents. Also disclosed are doses and methods of administration that show excellent effects when the fresh NK cells, cold-preserved NK cells and cryopreserved NK cells are used as pharmaceutical compositions for cellular therapy.
