Burkholderia cepacia MBA4 is a bacterium that degrades 2-haloacids by removing the halogen and subsequent metabolism of the product for energy. In this study, 2-DE, MS/MS, and N-terminal amino acid sequencing were used to investigate the protein expression profiles of MBA4 grown in a 2-haloacid (monochloroacetate, MCA) and in the corresponding metabolic product (glycolate). Glycolate was used as a control to eliminate the proteins induced by it. Five proteins were found to be up-regulated and five proteins were down-regulated in response to MCA. The differentially expressed proteins were examined, seven of them were identified by MS/MS and two of them were sequenced by Edman degradation. Our results definitely provide an insight for understanding the physiology of B. cepacia MBA4 in response to organohalide contaminated site.