Lysine lactylation (Kla) is a novel posttranslational modification, playing important roles in numerous biological processes, including transcription, metabolism, tumorigenesis and nervous system diseases. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), researchers have identified lactylation in many human organs and other species, but no research on lactylation in human hippocampi has been reported. In this study, we performed global profiling of lactylation in human hippocampi under normal physiological conditions, and we identified 2579 Class I (localization probabilities > 0.75) lactylated sites in 853 proteins. Bioinformatics analysis showed that lactylated proteins were mainly located in the cytoplasm and involved in metabolic pathways and some neurological diseases. Compared with those reported lactylation databases, 434 lactylated proteins were identified for the first time in this study. Our work expands the database on human lactylation and helps advance the study on lactylation function and regulation under physiological and pathological conditions.