Litopenaeus vannamei contains abundant phospholipids (PL), especially rich in polyunsaturated fatty acids (PUFA), which was easy to be hydrolyzed during storage and to produce free fatty acids (FFA). The increase of FFA leads to flavor, color and other changes, which affects the quality of aquatic products. Therefore in recent years, some information regarding the changes of lipids in aquatic food during handling and storage has been reported, such as L. vannamei, Pacific white shrimp and so on. However, the limited information on PL hydrolysis mechanism during storage is available, which is mainly owing to the complex system of PL hydrolysis and the lower content of phospholipases and lipase in aquatic food than mammals and microbes. Fortunately, electrospray ionization tandem mass spectrometry (ESI-MS/MS) has demonstrated high accuracy and reproducibility in phospholipids analysis, which brings potentiality for the study of the PL hydrolysis. Thus, to better understand the hydrolysis mechanism in sea foods during storage, we established a model system of phospholipids hydrolysis for the first time, which avoided the barrier of the complex system. We had extraction and purification of the phospholipases and PC from L. vannamei, and measured the enzyme activity of purified phospholipases. What's more, a fast and efficient "shotgun" lipidomics strategy was applied to analyze the levels and changes of PL, lysophosphatides (LPL) in L. vannamei. And the content of free fatty acids was analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the phospholipases extracted from L. vannamei included lipase, phospholipase A₁ (PLA₁), phospholipase A₂ (PLA₂), phospholipase C (PLC) and phospholipase D (PLD), among them PLA₂ had the highest enzyme activity. And in the reaction system, the content of PC decreased from 516.45 to 146.14 mg/g. Meanwhile, FFA had a significant increase from 36.42 to 568.57 mg/g, and PUFA increased by 280.5 mg/g. Furthermore, the study showed the close correlation between PC hydrolysis and PLA₂ (R=0.91) by measuring the enzymes activities before and after hydrolysis reaction. Besides, PLA₁ also showed the relatively close relationship with the PC hydrolysis compared to PLA₂. These information, mentioned above, suggested that aquatic phospholipases can hydrolyze phospholipids and produce a series of hydrolyzates, mainly including LPL and FFA. In a word, this study indicated the hydrolysis mechanism of phospholipids in aquatic products was correlated with the activities of lipid hydrolytic enzymes during storage, and PLA₂ was crucial to the hydrolysis of PC, which laid a theoretical foundation for the aquatic food storage and phospholipids research. Therefore, the suppression of lipid hydrolysis and the study of the complete hydrolysis mechanism will be a means to maintain the quality of aquatic food stored in ice, which may be a significant research direction in the future.