Abstract: Root differential proteomics was employed in this study to explore the impact of different planting patterns (multiple cropping rotation (MR) and consecutive monoculture multiple cropping (MC) modes) on growth of flue-cured tobacco (Nicotina tobaccum L). The results showed that a total of 15 protein spots had significant expression difference; 14 protein spots were identified by LC-MS/MS analysis and database searching. Among which, the function of 11 proteins were identified, three proteins were related to the aroma formation, i.e. transketolase, monodehydroascorbate reductase(MDHA), O-methyltransferase-like protein were up-regulated under MR, it was helpful probably to enhance the tobacco quality. While under the MC treatment, they were down-regulated, and the tobacco quality could be worse. Phosphoglycerate kinase, mitochondrial import inner membrane translocase subunit Tim13, NBS-LRR(nucleotide binding site plus leucine-rich repeat), DnaJ protein, Vesicle trafficking protein Sly1, Ribosomal protein-like protein were involved in energy metabolism, plant antioxidation reaction, translocation of solute, metabolism of nucleic acid, respectively. They were very important for the growth development of tobacco and all up-regulated under MR treatments, probably increasing the yield and improving the quality of tobacco, and gaining better economic benefit. They were all down-regulated under MC, which may cause weak growth, low yield and poor quality of tobacco.