Phosphorylation of myosin regulatory light chain (MRLC) can regulate muscle contraction and thus affect actomyosin dissociation and meat quality. The objective of this study was to explore the mechanism by how MRLC phosphorylation regulates actomyosin dissociation and thus develop strategies for improving meat quality. Here, the phosphorylation status of MRLC was modulated by myosin light chain kinase and myosin light chain kinase inhibitor. MRLC phosphorylation at Ser17 decreased the kinetic energy and total energy of actomyosin, thus stabilized the structure, facilitating the interaction between myosin and actin; this was one possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation. Moreover, MRLC phosphorylation at Ser17 was beneficial to the formation of ionic bonds, hydrogen bonds, and hydrophobic interaction between myosin and actin, and was the second possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation.