This study was conducted to evaluate the efficacy of a novel protease on egg production performance and fecal nutrient retention in laying hens fed corn- or wheat-based diet. Fifty-six Hy-Line Brown hens at 37 wk of age were fed two diets based on either wheat or corn, supplemented or not with protease at 30,000 NFP/kg. The diets were formulated with a 4 % reduction in protein and digestible amino acid compared to the breeder nutrient recommendations to reveal the potentials of protease. Each treatment included 14 replicate cages with 1 hen per cage. The study lasted 16 weeks for the measurement of production performance, and fecal nutrient retention during the last three days. Results showed that hens fed wheat-based diet had increased (P = 0.002) fecal nitrogen retention but less favorable (P = 0.095) feed efficiency than hens fed corn-based diet. The supplementation of protease, independently of diet type, increased egg production (+2.5 points, P = 0.005), fecal retention of dry matter (+3 points, P = 0.008) and AMEn (+128 kcal/kg, P <
0.0001). The present study demonstrated the efficacy of protease in terms of egg production as well as fecal nutrient retention in laying hens, highlighting the potentials for including protease in the nutritional programs of laying hens without compromising production performance.