A gene named zenc, encoding a zearalenone lactonase from Neurospora crassa, was over-expressed in Pichia pastoris. The zenc gene is 888-bp in length, encoding a 295-residue polypeptide. Purified ZENC has maximal activity at pH 8.0 and 45 °C, and is highly stable at pH 6.0-8.0 for 1 h at 37 °C. The activity of the secreted enzyme in shaken-flask fermentation was 40.0 U/ml. A high-density fermentation of the ZENC-producing recombinant strain was performed in a 30-l fermenter and the maximal enzyme activity reached 290.6 U/ml. The Km, Vmax and specific activity toward zearalenone are 38.63 μM, 23.8 μM/s/mg and 530.4 U/mg, respectively. ZENC can resist metal ions and inhibitors to some extent. We applied the enzyme into three different kinds of animal feed. On addition of ZENC (800 U) to distillers dried grains with solubles (DDGS), maize by-products and corn bran (25 g), the concentration of zearalenone was reduced by 70.9%, 88.9% and 94.7% respectively. All these properties of ZENC are promising for applications in the animal feed and food industries.