Background <p>Metabolism-induced post-translational modifications (PTMs) are critical for the regulation of cellular activities. As important types of modifications, lactylation and acetylation play essential roles in health and disease. With the development of lactylation research, its regulatory mechanism has been revealed to be highly similar to that of acetylation.</p> Main body <p>Most lactylated proteins are also acetylated. They share lysine modification sites and exert similar regulatory functions in gene transcription, DNA repair, signal transduction, autophagy, and metabolism, although certain differences exist. Both lactylation and acetylation regulate protein functions by affecting protein stability, enzyme activity, liquid-liquid phase separation, and crosstalk with other modifications. More importantly, the high similarity between their regulatory mechanisms brings challenges to their specific research and raises previously overlooked questions for published acetylation studies.</p> Conclusion <p>This review discusses the regulatory mechanisms, functional differences, and research dilemmas of lactylation and acetylation.</p>