Lactylation, a post-translational modification involving the addition of a lactyl group to lysine residues, has emerged as a critical regulatory mechanism in both health and disease. This review highlights the dual role of lactylation as a "double-edged sword," influencing cellular processes such as energy metabolism, inflammation, and tumor progression. Lactate, a key player in this modification, serves as both a metabolic substrate and a signaling molecule, facilitating energy production and intercellular communication. Recent studies have revealed that lactylation of histones and non-histones is involved in various physiological and pathological processes, including neural differentiation, osteogenesis, and cancer development. The mechanisms of lactylation involve enzymes such as acyltransferases, deacylases, and readers that regulate the modification and its downstream effects. Lactylation interacts with other post-translational modifications, such as acetylation, and plays a role in epigenetic regulation, immune responses, and metabolic reprogramming. In diseases like cancer, fibrosis, and inflammation, lactylation modulates gene expression, cell proliferation, and immune cell function. Targeting lactylation through drugs that inhibit lactate transporters (e.g., MCT1, MCT4, LDHA) or enzymes involved in lactylation (e.g., HATs, HDACs) shows promise for therapeutic applications. Overall, understanding lactylation's role in health and disease provides new insights into potential therapeutic strategies for various conditions.Lactylation, a post-translational modification involving the addition of a lactyl group to lysine residues, has emerged as a critical regulatory mechanism in both health and disease. This review highlights the dual role of lactylation as a "double-edged sword," influencing cellular processes such as energy metabolism, inflammation, and tumor progression. Lactate, a key player in this modification, serves as both a metabolic substrate and a signaling molecule, facilitating energy production and intercellular communication. Recent studies have revealed that lactylation of histones and non-histones is involved in various physiological and pathological processes, including neural differentiation, osteogenesis, and cancer development. The mechanisms of lactylation involve enzymes such as acyltransferases, deacylases, and readers that regulate the modification and its downstream effects. Lactylation interacts with other post-translational modifications, such as acetylation, and plays a role in epigenetic regulation, immune responses, and metabolic reprogramming. In diseases like cancer, fibrosis, and inflammation, lactylation modulates gene expression, cell proliferation, and immune cell function. Targeting lactylation through drugs that inhibit lactate transporters (e.g., MCT1, MCT4, LDHA) or enzymes involved in lactylation (e.g., HATs, HDACs) shows promise for therapeutic applications. Overall, understanding lactylation's role in health and disease provides new insights into potential therapeutic strategies for various conditions.