In this study, iTRAQ analysis and bioinformatics analysis were used to reveal the changes in key proteins induced by different concentrations of petroleum hydrocarbons during the biodegradation of petroleum hydrocarbons in Pseudomonas aeruginosa P6. Sixty-three proteins were identified as differentially expressed proteins, and all of them were strongly associated with the cellular processes related to the biodegradation of petroleum hydrocarbons. The results further showed that among the differentially expressed proteins, 3 chemotaxis-related proteins, 10 terminal oxidation of short-chain alkane-related proteins, and 13 transmembrane transport-related proteins were down regulated, while 1 uptake of petroleum hydrocarbon-related protein, 3 terminal oxidation of long-chain alkane-related proteins, 4 dehydrogenation-related proteins, 12 beta-oxidation-related proteins, and 2 metabolisms of acyl-CoA-related proteins were up regulated. These results indicated that during the biodegradation of petroleum hydrocarbons in P. aeruginosa P6, the activity of chemotaxis, the terminal oxidation of short-chain alkanes, and transmembrane transport decreased, while the activity of the uptake of petroleum hydrocarbons, the terminal oxidation of long-chain alkanes, dehydrogenation, beta-oxidation, and the metabolism of acyl-CoA increased under the 20,000 mg/L petroleum hydrocarbon condition compared with the 500 mg/L petroleum hydrocarbon condition. The findings revealed changes in the key proteins and the corresponding cellular process of the biodegradation of petroleum hydrocarbons in P. aeruginosa P6 under high and low concentrations of petroleum hydrocarbons and provided references for future studies.