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Polyamide4 (PA4) is a bioplastic with superior properties and high biodegradability. Although some PA4-degrading microorganisms have been isolated from various environments, enzymes that play a role in PA4 degradation are yet to be identified. In this study, we isolated a novel PA4-degrading microorganism, Pseudoxanthomonas sp. TN-N1. This strain showed a clear zone surrounding the colony on an agar plate with the medium containing PA4 powder. The PA4-degrading activity using the supernatant of the culture medium was maximum when the strain was cultured in an appropriate medium containing 0.5% (w/v) PA4 at pH 7.0, and 30 °C for 3 days. Furthermore, we purified an enzyme that hydrolyzes the amide bonds of PA4 into ?-aminobutyric acid oligomers (2–4 mers) from the supernatant of the culture medium using anion exchange and gel permeation column chromatography. However, the enzyme exhibited no protease activity for casein, and it consisted of monomers or dimers with a molecular mass of 90 kDa. The PA4-degrading activity was optimum at pH 7.5 and 35 °C, and more than 80% of the enzyme activity was retained in the pH range of 6.0– 7.0 and temperature below 35 °C. In addition, PA4-degrading activity was significantly inhibited by ethylenediaminetetraacetic acid treatment. In our opinion, this is the first report of an enzyme exhibiting PA4-degrading activity. Our findings will contribute to a better understanding of the mechanism of PA4 degradation by microorganisms.