The objective of this study was to investigate the effects of xylanase supplementation on gut hormone production in newly weaned piglets. In experiment 1, 32 female Camb12 weaned piglets (8.8 ± 1.38 kg, mean ± SD) were randomly assigned to 1 of 2 diet groups in period 1 (0–2 wk postweaning; P1), a control diet (Co) or the same diet supplemented with xylanase (XS). During period 2 (2–6 wk postweaning; P2) half the pigs were kept on their original diet while the rest were swapped onto the other diet, resulting in 4 groups. At the end of P2 the pigs were culled by electrical stun and exsanguination at which point blood samples were collected using EDTA coated tubes. The samples were centrifuged at 3000 g for 10 min at 4°C and the plasma was aliquoted and stored at −80°C for further analysis. A Human Metabolic Hormone Milliplex HMHMAG-34K kit (Merck Millipore) was used to assess the concentrations of PYY, PP, Insulin, C-Peptide and GIP (total) in the plasma. In experiment 2, 16 female Camb12 weaned piglets (9.2 ± 0.95 kg) were assigned to the same 2 diet groups as in experiment 1 (Co and XS). After P1, the pigs were culled and blood samples taken as in experiment 1; however, protease inhibitors were added to the collection tubes so extra gut hormones could be analyzed [amylin (active), ghrelin (active) and GLP-1 (active)]. The data were tested for normality and analyzed with the appropriate parametric or non-parametric test (one/two-way ANOVA or Kruskal-Wallis), significance was accepted at P < 0.05. In experiment 1, XS in P2 significantly decreased plasma PYY concentrations (P = 0.008). In experiment 2, XS significantly decreased plasma GIP (P = 0.027) and GLP-1 (P = 0.002) concentrations in P1. GIP and GLP-1 are known as incretin hormones that affect pancreas function. In conclusion, xylanase supplementation to newly weaned piglets affects the production of certain gut hormones. The effect was dependent upon the timing of supplementation as different hormones were affected between P1 and P2.Further trials are needed to investigate whether there are longer-term effects when the piglets are grown to a commercial slaughter weight.