This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, Achromobacter sp. strain C-1, from an industrial area (Cubatão, Brazil). Achromobacter sp. is a non-motile, strictly aerobic, gram-negative, short-rod or coccobacillary bacterium, which can occur singly, in pairs or in clusters. 16S rRNA gene sequence analysis revealed that Achromobacter sp. strain C-1 belongs to the gamma group of Proteobacteria, with 99% similarity to 16S rRNA gene sequences of Achromobacter xylosoxidans. Strain C-1 can grow aerobically on a number of aromatic compounds, such as phenol, catechol, m-cresol and o-cresol. In particular, it can degrade up to 600 mg/L of phenol at 37°C. In order to understand the phenol degradation pathway used by strain C-1, phenol or glucose cultured C-1 proteomes were comparatively analyzed with two-dimensional SDS-polyacrylamide gel (2D SDS PAGE). Nine protein spots were exclusively induced from phenol-cultures of strain C-1. Of these 9 spots, three phenol-degrading enzymes (phenol degradation meta-pathway protein, hydroxymuconic semialdehyde dehydrogenase and 4-hydroxy-2-oxovalerate aldolase) were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides, which suggests that strain C-1 degrades phenol via the β-ketoadipate pathway. Analysis of the metabolite produced from phenol proved that this enzyme is a catechol 2,3 dioxygenase, which is able to utilize phenol as a substrate. These results suggest that comparative proteomic analysis of biodegrading bacteria cultures under different conditions may be a useful initial step toward the elucidation of an aromatic compound degradation pathway and the strain C-1 could be an excellent candidate for the biotreatment of phenol-containing industrial wastewaters.