The impacts of cold stress (4 ℃ for 0 h, 12 h, 24 h, 36 h and 48 h, respectively) on the components, structural and physical properties of myofibrillar protein (MP) gel from Procambarus clarkii were investigated. The physicochemical analysis indicated the secondary and tertiary structure of MP were unfolding to different degrees after cold stress when compared to the control. The MP gel hardness reached a maximum when the cold stress reached 24 h. Furthermore, the quantitative proteomics results indicated that 20 up-regulated differentially abundant proteins (DAPs) were detected in 24 h when compared to control, specifically include myosin light chain 1 (MLC1) and skeletal muscle actin 6. Additionally, the combined analysis confirmed that MLC1 and skeletal muscle actin 6 might play key roles in hardening shrimp meat under cold stress. The results could provide a theoretical reference for the changes in crayfish muscle quality during cold chain transportation.