Vibrio parahaemolyticus is a kind of food-borne pathogenic bacterium, which can seriously infect food, especially seafood causing gastroenteritis and other disease. We studied the global proteome responses of V. parahaemolyticus under cold stress by nano-liquid chromatography-tandem mass spectrometry to improve the present understanding of V. parahaemolyticus proteomics events under cold stress. A total of 1151 proteins were identified and 101 proteins were differentially expressed, of which 69 were significantly up-regulated and 32 were downregulated. Functional categorization of these proteins revealed distinct differences between cold-stressed and control cells. These proteins were grouped into 21 functional categories by the clusters of orthologous groups (COG) analysis. The most of up-regulated proteins were functionally categorized as nucleotide transport and metabolism, transcription, function unknown, and defense mechanisms. These up-regulated proteins play an important role under cold stress.