Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O-2, leading to formation of piperonylic acid and H2O2. The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species.