The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post {YHV} infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is {MLCK} pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of {YHV} and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of {MLCK} inhibitors prior to {YHV} challenge resulted in dose-dependent elevation in quantity of YHV-positive {GC} and cytoplasmic {YHV} protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after {YHV} infection in shrimp and that inhibition of the phosphorylation leads to increased {YHV} replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the {MLCK} activation pathway may lead to new strategies in development and implementation of therapy for {YHV} infections in shrimp.