Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE_17390) and putative small catalase gene (AOLE_09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.