Protein acylation is a type of protein post-translational modification. Novel acylations except for acetylation, have been expanded successively in recent years. Histone acylation can directly modulate the packaging of chromatin either by altering the net charge of histone molecules or by altering inter-nucleosomal interactions, thereby promoting the regulation of transcription in the nucleus and the gene expression; besides, acylation can regulate the structure and function of non-histone protein, which changes in protein interactions between its binding partners, widely participating in a variety of cellular and molecular biological regulation. Exercise is one of the most critical factors that affect protein acylation, it not only enhances histone acylation level and involves in the regulation of gene expression, but also maintains tissue and cell metabolic homeostasis by regulating the histone acetyl transferases/histone deacetylases. Exercise modulates protein acylation through two pathways. On one hand, exercise regulates substance metabolism to change the level of metabolite in the human body, which provides abundant acyl-donors for acylation, such as acetyl-coenzyme A, succinyl-coenzyme A and lactoyl-coenzyme A. The acyl-coenzyme A comes from the intermediate products of glucose, fatty acids and amino acids metabolism in the body. Exercise-induced enhanced metabolism, increased tricarboxylic acid cycle rate, changes in energy content and enzyme activity all affect the concentration of acyl-CoA, thereby affecting the overall acylation level in a more three-dimensional and multi-dimensional manner. On the other hand, dramatic redox reactions and changes in kinase activity during exercise can also change the expression and activity of deacetylases such as the sirtuins family, regulating the balance of acylation/deacylation. Redox reactions in the intracellular surge during exercise cause an increase in the NAD⁺/NADH ratio, and can clearly activate sirtuins. In addition, altered levels of active factors and kinases can also promote gene and protein expression of enzymes and regulate deacylation. Briefly, the regulation of exercise on protein acylation is a new mechanism for exercise that improves metabolism, promotes health and prevents chronic diseases, relevant research work is still in its infancy and deserves special focus.