This review explores the role of protein lactylation, a novel post-translational modification mediated by lactate, in various physiological and pathological processes. Lactate, traditionally viewed as a metabolic waste product, has been discovered to act as both a substrate and a product, playing a crucial role in energy metabolism, signal transduction, and immune regulation. The review highlights the significance of lactylation in human health, including neural and osteogenic differentiation, and its involvement in diseases such as tumors, fibrosis, and inflammation. Key mechanisms of lactylation, including the involvement of specific enzymes and the crosstalk with other post-translational modifications, are discussed. The review also examines the impact of lactylation on cellular development, inflammation, brain diseases, fibrosis, and tumors, providing insights into its potential as a therapeutic target. Finally, the authors suggest that targeting lactate and its transporters, as well as lactylation enzymes, could be a promising approach for treating various diseases.This review explores the role of protein lactylation, a novel post-translational modification mediated by lactate, in various physiological and pathological processes. Lactate, traditionally viewed as a metabolic waste product, has been discovered to act as both a substrate and a product, playing a crucial role in energy metabolism, signal transduction, and immune regulation. The review highlights the significance of lactylation in human health, including neural and osteogenic differentiation, and its involvement in diseases such as tumors, fibrosis, and inflammation. Key mechanisms of lactylation, including the involvement of specific enzymes and the crosstalk with other post-translational modifications, are discussed. The review also examines the impact of lactylation on cellular development, inflammation, brain diseases, fibrosis, and tumors, providing insights into its potential as a therapeutic target. Finally, the authors suggest that targeting lactate and its transporters, as well as lactylation enzymes, could be a promising approach for treating various diseases.