The objectives of this study were to investigate the effects of alanyl-glutamine (Ala-Gln) dipeptide supplementation on the intestinal mucosa barrier in weaned piglets challenged by lipopolysaccharide (LPS). A total of 96 piglets were arranged in a 2 × 2 factorial design and randomly divided into four groups. The main effects were LPS challenge (0 or 100 μg kg^-1 body weight) and diets (0.62% Ala or 0.5% Ala-Gln). The results showed that LPS challenge significantly increased the crypts depth and decreased the villi height of small intestine in the piglets (P < 0.05), whereas, dietary Ala-Gln supplementation increased the villi height of small intestine (P < 0.05). The LPS challenge decreased the mRNA expressions of epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) in the jejunal mucosa (P < 0.05), and Ala-Gln supplementation promoted the mRNA expressions of EGFR and IGF-1R. In addition, Ala-Gln supplementation also increased the mRNA and protein expressions of Claudin-1 and zonula occludens protein-1 (P < 0.05), decreased the number of goblet cells in the jejunal and ileal epithelium (P < 0.05) and the mRNA expressions of interleukin-1β and interleukin-8 in the jejunal mucosa (P < 0.05). There was no significant interaction between Ala-Gln supplementation and LPS challenge in most parameters except for the concentrations of the jejunal mucosa immunoglobulins (P < 0.05). The results indicated that Ala-Gln supplementation was beneficial to strengthen the intestinal mucosal barrier in piglets and could partially attenuate the adverse effects caused by LPS challenge.