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Fumonisins (FB) are among the most frequently detected mycotoxins in feedstuffs and finished feed, and recent data suggest that the functions of the gastrointestinal tract (GIT) in poultry species might be compromised at doses ranging from 10 to 20 mg/kg, close to field incidences and below the US and EU guidelines. Strategies are therefore necessary to reduce the exposure of poultry to FB. In the present study, we assessed the efficacy of fumonisin esterase FumD (EC 220.127.116.11, commercial name FUMzyme®) to cleave the tricarballylic acid side chains of FB, leading to the formation of non-toxic hydrolyzed fumonisins in the GIT of broiler chickens. Broiler chickens were fed for 14 d (7 to 21 d of age) 3 different diets (6 birds/cage, 6 cages/diet), i) control feed (negative control group), ii) feed contaminated with 10 mg FB/kg (FB group), and iii) feed contaminated with 10 mg FB/kg and supplemented with 100 units of FUMzyme®/kg (FB+FUMzyme® group). To determine the degree of reduction of FB in the GIT, 2 characteristics were analyzed. First, the sphinganine-to-sphingosine ratio in the serum and liver was determined as a biomarker of effect for exposure to FB. Second, the concentration of fumonisin B1 and its hydrolyzed forms was evaluated in the gizzard, the proximal and distal parts of the small intestine, and the excreta. Significantly reduced sphinganine-to-sphingosine ratios in the serum and liver of the FB+FUMzyme® group (serum: 0.15 ± 0.01; liver: 0.17 ± 0.01) compared to the FB group (serum: 0.20 ± 0.01; liver: 0.29 ± 0.03) proved that supplementation of broiler feed with FUMzyme® was effective in partially counteracting the toxic effect of dietary FB. Likewise, FB concentrations in digesta and excreta were significantly reduced in the FB+FUMzyme® group compared to the FB group (P < 0.05; up to 75%). FUMzyme® furthermore partially counteracted FB-induced up-regulation of cytokine gene expression (IL-8 and IL-10) in the jejunum. The FB group showed significantly higher gene expression of IL-8 and IL-10 compared to the negative control group (IL-8: fold change = 2.9 ± 1.1, P < 0.05; IL-10: fold change = 3.6 ± 1.4, P < 0.05), whereas IL-8 and IL-10 mRNA levels were not significantly different in the FB+FUMzyme®® group compared to the other 2 groups. In conclusion, FUMzyme® is suitable to detoxify FB in chickens and maintain gut functions.