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The study was conducted to determine the effects of high levels of phytase on growth performance, nutrient digestibility, phytate breakdown, and expression of mucosal tight junction and nutrient transporter genes in weanling pigs. A total of 128 barrows were penned in groups of four and used in a randomized completely block design and assigned to four treatments for a 28-d study. A two-phase feeding was implemented (phase 1: day 1 to 14; phase 2: day 15 to 28). The diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8.1 to 7.1 g/kg Ca and 6.5 to 6.8 g/kg P; negative control [NC]: 6.6 to 5.5 g/kg Ca and 5.6 to 5.3 g/kg P) from phase 1 to phase 2, respectively. NC diets were supplemented with phytase at 0 (NC), 1,500 (NC + 1,500), or 3,000 (NC + 3,000) phytase units (FTU)/kg. Blood was collected after fasting (day 27) or feeding (day 28) for the measurement of plasma inositol concentrations. On day 28, two pigs per pen were euthanized. Duodenal-jejunal and ileal digesta samples and feces were collected to determine inositol phosphates (InsP3-6) concentrations. Phytase supplementation increased the body weight on days 14 and 28 (P < 0.05). Average daily gain and feed efficiency compared with NC were increased by phytase with the majority of its effect in phase 1 (P < 0.05). The apparent ileal digestibility and apparent total tract digestibility of P were increased in piglets fed phytase-supplemented diets (P < 0.01) compared with NC piglets. Disappearance of InsP6 and total InsP3-6 up to the duodenum-jejunum, ileum, and in feces was increased by both phytase application rates (P < 0.01). Plasma concentrations of myo-inositol were higher (P < 0.001) in the phytase-supplemented diets than PC and NC diets, irrespective of whether pigs were fed or fasted. Expression of claudin 3 was higher in pigs fed both phytase-supplemented diets in the duodenum and jejunum compared with PC and NC. Mucin 2 expression was lower in the ileum of NC + 3,000 fed piglets compared with PC (P < 0.05), whereas expression of GLUT2 (solute carrier family 2-facilitated glucose transporter member 2) was increased (P < 0.05) by the NC + 3,000 treatment in all sections. In summary, high phytase supplementation increased the growth performance of nursery pigs. The increased expression of GLUT2 by phytase may indicate an upregulation of glucose absorption from the intestine by phytase.