This study focused on the protein expression of a Microbacterium sp. strain that utilized various concentrations of benzo(a)pyrene (BaP) as the sole source of carbon and energy under anaerobic conditions. A total of 1539 protein species were quantified by isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS. GO, COG, and pathway enrichment analysis showed that most proteins demonstrated catalytic and binding functions and were mainly involved in metabolic processes, cellular processes, and single-organism processes. Sixty-two proteins were found in their abundances in BaP-stress conditions different from normal conditions. These proteins function in the metabolic pathways; the biosynthesis of secondary metabolites, the biosynthesis of antibiotics, microbial metabolism in diverse environments, carbon metabolism, and the biosynthesis of amino acids were markedly altered. Furthermore, enoyl-CoA hydratase was proposed to be a key protein during BaP removal of the Microbacterium sp. strain. This study provides a powerful platform for the further exploration of BaP removal, and the differentially expressed proteins provide insight into the mechanism of the BaP removal pathway.