The freshness of Pacific white shrimp (Litopenaeus vannamei) stored for 0, 2, 4, 6, 8, and 10 days under vacuumpacking at 4°C was assessed by physicochemical, sensory, and microbiological methods. Thiobarbituric acid, total volatile basic nitrogen, pH, total viable counts, and K values increased significantly (P < 0.05) with the storage time, whereas the texture profile indexes decreased significantly (P < 0.05). Furthermore, a proteomic approach of 2-dimensional gel electrophoresis coupled with MALDI-TOF/TOF identification was used to analyze the differentially expressed muscle proteins at the different storage times. All of the 3 identified proteins (arginine kinase, phosphopyruvate hydratase, and actin T², which were involved in muscle energy metabolism, glycolytic pathway in adenosine triphosphate generation, and organization of myofibril, respectively) were downregulated with the increase of storage time. Quantitative reverse transcription—polymerase chain reaction showed that messenger RNA levels of these proteins also decreased during storage time. Downregulation of the identified proteins may contribute to changes of flesh texture and color. This study has important implications for understanding the molecular mechanism of postmortem changes and for developing predictive models suitable for determining the freshness and shelf life of shrimp.