Abstract: 【Objective】Study the molecular mechanism of Ilex×koehneana ‘Emily Bruner' in response to low temperature stress.【Mothed】 We analyzed the proteomic differences in leaves among different temperature treatment(0 ℃, -8 ℃ and -16 ℃)by using 2-DE coupled with MALDI-TOF/TOF MS.【Result】 The 2-DE profile of the total protein in the leaves demonstrated that more than 1 000 protein spots were detected with good reproducibility in the range of molecular masses of 15-120 ku and pH 4-7. A total of 31 protein spots in low temperature treatments exhibited significant changes in abundance of less than 0.66-fold or more than 1.5-fold(P<0.05)compared to CK. After MALDI-TOF/TOF MS analysis, a total of 23 differentially accumulated protein spots were successfully identified. Among the 23 identified proteins, 20 proteins had assigned functions, and the other 3 were identified as novel with no assigned function. The 20 proteins with assigned functions were classified into 8 metabolic pathways and cellular processes, including protein and amino acid metabolism, carbohydrate metabolism, carbon fixation in photosynthetic organisms, lipid metabolism, photosynthesis, redox homeostasis, defense response, and nitrogen metabolism. The identifications of these differentially accumulated proteins indicates the presence of a specific different metabolic network in Ilex in response to cold stress.【Conclusions】These studies as a first step towards understanding the molecular mechanisms of low temperature resistance of Ilex, will provide the research basis for cultivating and screening relatively cold resistant varieties.