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l‐Theanine (N‐ethyl‐ l‐glutamine) is an analog of l‐glutamine and l‐glutamic acid, accounts for up to 50% of all free amino acids in green tea, and elicits an umami taste. As l‐theanine also shows various physiological activities including immune response‐modifying activities, it is expected to be an excellent health‐promoting phytochemical agent. To know the influences of l‐theanine on the human innate immune response, we investigated the effect of l‐theanine on the superoxide anion (O2−)‐generating system of leukocytes using U937 cells. The O2−‐generating system in leukocytes consists of membrane cytochrome b558 protein (a complex of p22‐phox and gp91‐phox proteins) and cytosolic p40‐phox, p47‐phox, and p67‐phox proteins. Addition of 500 μM l‐theanine caused remarkable enhancement of the all‐trans retinoic acid (ATRA)‐induced O2−‐generating activity (to ~470% of ATRA‐treated cells), but not l‐glutamine and l‐glutamic acid. Semiquantitative RT‐PCR showed that the transcription level of gp91‐phox is significantly increased in ATRA and l‐theanine‐co‐treated cells. Chromatin immunoprecipitation revealed that l‐theanine enhances acetylations of Lys‐9 and Lys‐14 residues of histone H3 within the chromatin surrounding the promoter region of the gp91‐phox gene. Immunoblotting demonstrated that membrane cytochrome b558 proteins remarkably accumulate in ATRA + l‐theanine‐treated cells. These results suggested that l‐theanine brings about a remarkable accumulation of cytochrome b558 protein via upregulating the transcription of the gp91‐phox gene during leukocyte differentiation, resulting in marked augmentation of the O2−‐generating ability, which is one of the most important functions of leukocytes responsible for the innate immune system. [ABSTRACT FROM AUTHOR]