Natural killer-92 cells (NK-92 cells) need to be efficiently expanded by serum-free culture in vitro to meet clinical requirements. Fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of highly proliferating cells. This study optimized the medium (EM) for NK-92 cells by designing an experiment to expand cells efficiently. EM, an in-house designed chemically defined serum-free medium, was used as the basal medium. Fatty acids as additive ingredients were screened and optimized by the experimental design method. Three additives, arachidonic acid, myristic acid and palmitoleic acid, were screened; therefore, the optimized medium was named EM-FA. The total cell expansion of NK-92 cells in EM-FA was 72.61 +/- 11.95-fold on day 8, which was significantly higher than the 28.55 +/- 8.67-fold expansion in EM. To explore the mechanism by which fatty acids promote NK-92 cell expansion, the cell growth kinetics and metabolic characteristics in EM-FA were analyzed. The results showed that NK-92 cells in EM-FA were rapidly expanded while maintaining their cell phenotype and cytotoxicity and enhancing the oxygen consumption rate and mitochondrial function. Fatty acids promoted ATP production to elevate the energy flux for better cell expansion. This study developed an expansion strategy of NK-92 cells in vitro to facilitate their clinical application. Key points Arachidonic acid, myristic acid and palmitoleic acid in serum-free medium were optimized by experimental design to enable the rapid expansion of NK-92 cells in vitro. Fatty acids upregulated oxidative phosphorylation levels and improved the energy metabolism of NK-92 cells.