Lactate has diverse roles in the brain at the molecular and behavioral levels under physiological and pathophysiological conditions. This study investigates whether lysine lactylation (Kla), a lactate-derived post-translational modification in macrophages, occurs in brain cells and if it does, whether Kla is induced by the stimuli that accompany changes in lactate levels. Here, we show that Kla in brain cells is regulated by neural excitation and social stress, with parallel changes in lactate levels. These stimuli increase Kla, which is associated with the expression of the neuronal activity marker c-Fos, as well as with decreased social behavior and increased anxiety-like behavior in the stress model. In addition, we identify 63 candidate lysine-lactylated proteins and find that stress preferentially increases histone H1 Kla. This study may open an avenue for the exploration of a role of neuronal activity-induced lactate mediated by protein lactylation in the brain. Display omitted •Neural excitation increases lactate levels and lysine lactylation in the brain•Social defeat stress increases brain lactate and lactylation levels chronically•Stress-associated neural excitation stimulates histone H1 lactylation•The increased histone H1 lactylation is correlated with decreased social behavior Hagihara et al. find that lysine lactylation in brain cells is regulated by systemic changes in lactate levels, neural excitation, and social defeat stress. Sixty-three lactylated proteins are identified in the mouse brain. They provide evidence for lactylation in the brain and its regulation by neural-activity-induced lactate.