A novel chemically defined medium, named KG medium, supplemented with N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), an acylhomoserine lactone (AHL) used as signalling molecules in Gram-negative bacterial cell-to-cell communication, as the sole source of carbon and nitrogen, was designed and successfully used for the enrichment and isolation of AHL-degrading bacteria. A 3-oxo-C6-HSL-degrading bacterium, 13sw7, was isolated from sewage after six enrichment transfers in the 3-oxo-C6-HSL-containing KG medium. On the basis of the almost complete 16S ribosomal DNA sequence, isolate 13sw7 was clustered with unculturable beta-proteobacteria. This study indicates that the AHL-containing KG medium is effective in isolating AHL-degrading bacteria, including those previously considered unculturable, from environmental sources. To the best of our knowledge, this is the first documentation of the isolation of an AHL-degrading proteobacterium from sewage.