Herein, to unveil the role of phosphorylation modifications in texture softening of shrimp muscle during refrigeration, the phosphorylation profiling of muscle structural proteins after chilling storage at 4 oC for 120h was comparatively investigated by phosphoproteomics. Totally, the unique 1255 differential abundance phosphopeptides (DAPPs), encompassing 2128 phosphorylated sites in 608 phosphoproteins, were identified and they were primarily classified into intracellular myofibril, cytoskeleton, and assembly regulating factors. GO and KEEG annotation analysis revealed that these DAPPs were dominantly enriched in ultrastructure assembly and myofilament contraction of sarcomere architecture, pathways of mTOR signaling, autophagy, and energy metabolism. Meanwhile, the profiling of concomitant dephosphorylation of kinases, phosphatases, and assembly regulating factors revealed the disassembly potential of sarcomeric architecture through SP and R/KxxS motifs. Moreover, the predominate nodes in protein-protein interaction (PPI) network included mitogen-activated protein kinase 14, ubiquitin-ribosomal protein eL40 fusion protein, cAMP-dependent protein kinase catalytic subunit, heat shock protein, and myosin light chain kinase; meanwhile, these proteins showed significantly high correlation with texture deterioration, indicating their potential as target regulators for intervening the muscle softening process. Collectively, this study, for the first time, deciphered the undefined mechanisms governing muscle softening of shrimp L. vannamei during refrigeration from the phosphorylation-dependent perspective. Furthermore, these insights might provide valuable alternatives for developing target strategies against muscle softening by manipulating phosphoproteins.