The myosin regulatory light chain 2 (MLC2) is a crucial regulator of myosin activity. Its phosphorylation, mediated by various kinases, plays a vital role in maintaining normal physiological functions in skeletal muscle, myocardium, smooth muscle, and nonmuscle cells. Moreover, MLC2 has been implicated in the development of many cancers through its phosphorylation. An increasing number of studies have shown that MLC2 may influence tumor progression by modulating cancer cell growth, migration, invasion, apoptosis, and autophagy. In this paper, we provide a concise overview of the phosphorylation regulatory mechanisms of MLC2 and its roles in both physiology and tumorigenesis. Furthermore, this study proposes potential directions for future research.