Abstract: iTRAQ proteomics approach was developed to investigate differentially expressed proteins in Pseudostellariae Radix from different habitats. The extracted proteins were digested by FASP, identified with iTRAQ coupled with LC-MS/MS and then analyzed by Protein Pilot 5.0 search engine, through the comparison of relative quantitative protein in search of differentially expressed proteins. The analysis of differentially expressed proteins was conducted using GO (gene ontology), KEGG and STRING. A total of 3 775 protein are detected, among them, 3 676 proteins are provided quantitative information, of which 54 proteins are up-regulated and 86 are down-regulated in traditional fields of Pseudostellariae Radix. 44 significantly differential expressed proteins are found, which are classified into nine categories, such as Heat shock proteins, Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, RuBisCO large subunit-binding protein, Chaperone protein, Luminal-binding protein. The results indicated that catabolic, carbohydrate metabolism and respond to stress of Oxidoreductases and Transferases in traditional fields of Pseudostellariae Radix are stronger than other cultivated habitats of Pseudostellariae Radix, protein folding and respond to stress of Heat shock proteins, Isomerases, RuBisCO large subunit-binding protein, Chaperone protein, Luminal-binding protein are weaker, catabolic of Hydrolases and Lyases are no difference between traditional fields of Pseudostellariae Radix and other cultivated habitats. ADG1 and TKTA are the important proteins to regulate sucrose in Pseudostellariae Radix from different habitats, while MFP2 accommodate the fatty acids. This work can provide the basic information for exploring the cause of different habitats for Pseudostellariae Radix secondary metabolites and the mechanism of proteins for quality forming process.