Background: Dysfunction of tight junction integrity is associated with decreased nutrient absorption and numerous gastrointestinal diseases in humans and piglets. Although L-glutamine has been reported to enhance intestinal-mucosal mass and barrier function under stressful conditions, in vivo data to support a functional role for L-glutamine on intestinal tight junction protein (TJP) expression in weanling mammals are limited. Objective: This study tested the hypothesis that glutamine regulates expression of TJPs and stress-related corticotropinreleasing factor (CRF) signaling in the jejunum of weanling piglets. Methods: Piglets were reared by sows or weaned at 21 d of age to a corn and soybean meal-based diet that was or was not supplemented with 1% L-glutamine for 7 d. Growth performance, intestinal permeability, TJP abundance, and CRF expression were examined. Results: Weaning caused increases (P < 0.05) in intestinal permeability by 40% and in CRF concentrations by 4.7 times in association with villus atrophy (P < 0.05). Western blot analysis showed reductions (P < 0.05) in jejunal expression of occludin, claudin-1, zonula occludens (ZO) 2, and ZO-3, but no changes in the abundance of claudin-3, claudin-4, or ZO-1 in weanling piglets compared with age-matched suckling controls. Glutamine supplementation improved (P < 0.05) intestinal permeability and villus height, while reducing (P < 0.05) jejunal mRNA and protein levels for CRF and attenuating (P < 0.05) weanling-induced decreases in occludin, claudin-1, ZO-2, and ZO-3 protein abundances. Conclusion: Collectively, our results support an important role for L-glutamine in regulating expression of TJPs and CRF in the jejunum of weanling piglets. © 2015 American Society for Nutrition.