Abstract: To understand the differential expression proteins and their functional mechanisms in root system of seeding stage high potassium flue-cured tobacco under low potassium stress, root proteins were extracted from seedling stage high potassium flue-cured tobacco variety HKDN-5 cultivated in low potassium and normal potassium nutrient solutions. After enzymolysis LC-MS and label-free protein quantification technique were used to identify and analyze the differential expression of proteins. The results showed that there were 681 differentially expressed proteins in the low potassium group compared with the normal potassium group, 359 proteins were down regulated and 322 proteins were up regulated. Differential expression proteins were mainly involved in lipid metabolism, secondary metabolites biosynthesis, carbohydrate metabolism and genetic information processing. The highest number of down regulated proteins was identified with the function of post translational modification, protein transformation, and chaperone related functions, the up regulated proteins were mainly the general functional related proteins. The proteins with large change folds were mainly related to metabolic and stress response processes. The above results indicated that the differential proteins produced by low potassium stress may participate in related processes such as substance metabolism and stress responses.