Burkholderia caribensis MBA4 is a Gram-negative soil bacterium with the ability to utilize haloacetic acids (HAAs) as carbon source. A single dehalogenase (Deh4a) was isolated from monobromoacetate grown MBA4. The structural gene of Deh4a has been identified and characterized. An operon containing a dehalogenase gene (deh4a) and a permease gene (deh4p) is negatively regulated. At least one DNA binding protein was found to bind on the upstream non-coding region of deh4a when HAA was absent. In this work, a putative regulator that controls the expression of deh4a was identified by Tn5 transposon mutagenesis. A derivative of MBA4 containing a kanamycin resistant gene (kan) driven by deh4a promoter was constructed and used for genome transposon mutagenesis. The derivative performs kanamycin resistant (KmR) while using monochloroacetic acid (MCA) as growth substrate and kanamycin sensate (KmS) in medium with pyruvate. KmR mutants in un-induced condition were isolated and the Tn5 insertion site was determined. A gene encoding a putative transcription regulator was identified. Disruption of this gene in MBA4 relieves the repression of the haloacid operon in the absence of HAAs. Moreover, production of this putative regulator in the mutant suppresses the transcription of deh4a in un-induced condition.