In addition to being an essential amino acid in protein synthesis, threonine is an integral component of gut function. To verify the effects of l-threonine on gut function, Babcock Brown layers (n = 960; 40 wk of age) were allocated to 5 dietary treatment groups, each of which included 6 replicates of 32 hens. Each group received the same basal diet formulated with corn, peanut meal, and crystalline amino acids. l-Threonine was added to the basal diet at 0 (control), 0.1, 0.2, 0.3, and 0.4% for 8 wk to achieve 0.47 (NRC), 0.57, 0.67, 0.77, and 0.87% threonine, respectively. Expressions of jejunal and ileal mucin 2 mRNA were increased linearly by increasing l-threonine (P < 0.01). At 0.4% l-threonine, the concentrations of IgA antibody in the mucosa of the ileum increased linearly (P < 0.01). No differences attributable to treatment were found among groups in the activity of digestive enzymes in the jejunum or ileum. It was concluded that dietary threonine requirements as reported in current NRC recommendations are insufficient for modern commercial laying hens raised in summer climates. The results suggest that threonine might function as a nutrient immunomodulator in maintaining intestinal barrier function.