PURPOSE: The hippocampus has long been associated with cognition and memory function, the implications of lysine lactylation (Kla), a recently identified post-translational modification (PTM), in the role of the hippocampus remain largely unexplored. EXPERIMENTAL DESIGN: An LC-MS/MS bottom-up proteomics analysis of three human hippocampal tissue samples was applied to profile the lactylation map in human hippocampi under normal physiological conditions. RESULTS: We identified 2579 quantifiable Class I lactylated sites in 853 proteins, of which contained four types of modification motifs. Cellular localization analysis implies that a majority of lactylated proteins were distributed in the cytoplasm. Functional enrichment analysis showed that lactylated proteins were mainly involved in energy metabolic pathways. In addition, we found that the lactylation on histones exhibits a certain degree of conservation across different tissues. Compared with previously reported lactylation databases, 213 lactylated proteins were identified for the first time in this study. CONCLUSION AND CLINICAL RELEVANCE: The first global lactylated proteins atlas of human hippocampi was reported in this study. Our work provides a reliable foundation for further research on lactylation in the hippocampus under physiological conditions.