In this study, we used three cotton accessions‘Xinluzao No.33’, ‘Xinluzao No.36’ and ‘Xinluzao No.50’which were locally approved and widely used to explore protein changes resulted from seed deterioration in cotton. By accelerating aging at high tempreture and humidity（40 ℃，RH 100%），we obtained cotton seeds with different vigor levels.Proteomic of vigor deteriorated seed of ‘Xinluzao No.50’ analysised by two dimensional gel electrophoresis technology, and differential protein spots were identified by MALDITOFMS/MS mass spectrometry technology，to explicit change of proteome expression in the process of cotton seed vigor deterioration. The results showed: (1) among the three accessions, ‘Xinluzao 50’ displayed a linear decrease in seed vitality as the aging treatment extended. (2) Approximately 350 protein spots were observed in the 2DE gels within the area of pH47 and molecular weight from 14.4 kD to 97.4 kD. Among them, 30 were identified as differential protein spots with at least two fold change of expression levels and statistically significant difference at 99% level. (3) Using the method of MALDITOF/TOF, twentynine protein spots were successfully identified.Bioinfomatic analysis results indicated that we obtained 13 storage related proteins, two immunity related proteins, one lipid metabolism related protein, one energy metabolism related protein, two actins, two regulatory related protein, one secondary metabolites, one cell defense related protein, one secreted protein, and five proteins with unknown functions. The study identified 29 differentially expressed proteins probably are associated with cotton seed vigor deterioration.