Endo-xylanase hydrolysis of xylan releases xylo-oligosaccharides (XOS), which act as prebiotics in poultry by stimulating fibre-fermenting bacteria and enhancing short-chain fatty acid (SCFA) production in the caeca. This study hypothesized that a carbohydrase preparation would generate XOS in a substrate-dependent manner, and that in vitro release and in vivo caecal fermentation responses could be linked. Two broiler diets (wheat-soy or maize-soy) were tested ± a mixture of xylanase (560 TXU/kg) and β-glucanase (250 TGU/kg; Natugrain® TS, BASF SE, Germany) in a 2 × 2 factorial arrangement in vitro and in vivo. in vitro, diets were subjected to a two-step digestion (gastric and intestinal) to quantify XOS release (degree of polymerisation 2 – 5). in vivo, each treatment had eight replicates of 12 birds. Overall (d 0-35) growth performance was recorded, and caecal SCFA were measured at d 21 from four birds per replicate pen. in vitro, a diet × enzyme interaction was observed where enzyme addition increased XOS2 (P = 0.049) and XOS3 (P = 0.010) release in the wheat-soy diet but not in the maize-soy diet. XOS4 and XOS5 were absent in the maize-soy diet, whereas both increased with enzyme addition in the wheat-soy diet (P = 0.042 and 0.001, respectively; independent t-test), highlighting that wheat has more enzyme-susceptible xylans and more potential for enzyme-mediated prebiotic XOS generation. in vivo, an interaction was observed for d35 body weight (P = 0.013), with a greater enzymes response in birds fed the maizesoy diet than the wheat-soy diet. Enzyme supplementation improved overall FCR (P < 0.001) by 9.3 points in the wheat-soy diet and 6.8 points in the maize-soy diet. The wheat-soy diet led to greater total caecal SCFA level (P = 0.013) than those fed the maize-soy diet. Enzyme increased caecal butyric acid (P = 0.039) and tended to increase total SCFA (P = 0.082). In the wheat-soy diet, in vitro XOS2-XOS5 levels positively correlated with in vivo caecal SCFA production (P < 0.045), with XOS5 showing the strongest link (r = 0.523, P = 0.018). In conclusion, enzyme supplementation improved feed efficiency in both diets. Enhanced XOS release and caecal fermentation were more pronounced with the wheat-soy diet. Further research is needed to fully clarify how enzymemediated XOS release consistently translates into improved gut function and bird performance.