Previous research has reported that a switch in the biodegradation pathway of sodium benzoate (SB) by Pseudomonas putida ATCC 49451 could be initiated via a change in the initial substrate concentration. With SB up to 200 mg/L, the substrate was degraded via the ortho pathway. However, cells grown on high enough concentrations (≥300 mg/L) were shown indirectly to activate both the ortho and the meta pathways. It is anticipated that this pathway-switch could result in a variety of physiological changes within the cells. In this study, proteome analysis by means of 2-D gel electrophoresis and MALDI-TOF MS was used to investigate the physiological responses that occurred in P. putida when grown at high SB concentration. Proteome profiles of the cells harvested from cultures grown on 100 mg/L, and 800 mg/L of SB were compared after 2-D PAGE. Coefficients of correlation between biological and analytical replicates were 0.74 ± 0.03 and 0.85 ± 0.05, respectively. Based on Student's t-test with p value less than 0.05, differential expression of 18 protein spots (newly induced designated H1-H5, up-regulated designated U1-U10, and down-regulated designated D1-D3) were found in cells grown on the higher substrate concentration (800 mg/L) compared with those grown on 100 mg/L. The protein spots were identified using PMF or PSD method. Of particular significance were the four newly induced protein spots which were identified as key enzymes in the meta degradation pathway. Additionally, enzyme assay results showed that the activity of catechol 2,3- dioxygenase was only detected in cell lysates obtained from high SB concentration. To our knowledge, these results are the first to provide confirmation of the activation of the meta degradation pathway at high SB concentration directly at the protein level. Furthermore, the induction of stress response proteins and other adaptation phenomena in response to high SB concentration were also inferred based on the protein spots that were also identified.