Abstract: To investigate the effect of salt stress on protein expression of cotton seedling roots and enrich the mechanism of salt tolerance at molecular level, this research studied the differential proteomics of cotton seedling roots under salt stress. 1% NaCl was used to simulate salt stress,Gossypium hirsutum L.‘Zhongmian 69’was treated for 72 h, the material was grinded with phosphate buffer, and the proteins in seedling root were extracted with carrier-assisted trichloroacetic (TCA) precipitation. The two-dimensional electrophoresis was conducted. The differential protein expression profiles with high resolution and clear background were obtained. The mapping analysis showed that there were 36 protein spots in cotton seedling roots responded to salt stress, in which 16 spots were up-regulated and 20 spots were down-regulated. The 10 differentially expressed proteins were analyzed by mass spectrometry and were identified as homologous proteins, they were ascorbate peroxidase (APX), enolase, glutathione-S-transferase, quinone oxidoreductase, annexin AnxGb3, chloroplast Rubiso enzyme subunit binding protein alpha subunit, P23, fructokinase, alcohol dehydrogenase, adenine phosphoribosyltransferase- 1-subtype-1. The results showed that some of the cotton seedling root proteins appeared significant differential expression, which indicated that these proteins might participate in the signal and metabolism way of salt resistance.