Beta-alanine is an important amino acid involved in several metabolic reactions in the body. The study aimed to investigate the effect of β-alanine supplementation on intestinal development and the immune performance of weaned piglets. Thirty-two 21-day-old healthy weaned piglets (half female and half male; Duroc × Landrace × Yorkshire) with an initial body weight of 8.11 ± 0.21 kg were randomly divided into 4 groups with 8 replicates of 1 pig each. The control group was fed a basal diet and the three experimental treatment groups were fed diets supplemented with 300, 600 and 1,200 mg/kg β-alanine, respectively. The trial lasted 28 days and the diets fed were divided into 2 phases: the late lactation period (day 1 to 14) and the nursery period (day 15 to 28), during which the weaned piglets had free access to food and water. The regulatory effects of β-alanine were further investigated in vitro using organoids obtained from the jejunum of piglets. In vivo, the addition of β-alanine to the diet had no significant effect on the growth performance of weaned piglets (P> 0.05), but significantly reduced serum levels of immunoglobulin G (IgG) (P < 0.01), immunoglobulin M (IgM) (P= 0.005), and complement 3 (C3) (P= 0.017). The serum interleukin- 6 (IL-6) levels (P< 0.01) were significantly reduced in the 1,200 mg/kg treatment group. The addition of β-alanine increased ileal villus height, with the most significant effect at a concentration of 300 mg/kg (P= 0.041). The addition of 600 mg/kg β-alanine significantly up-regulated the expression of superoxide dismutase (SOD) activity (P= 0.020) and the zonula occludens-1 (ZO-1) gene (P= 0.049) in the jejunum. Diets supplemented with 300 mg/kg βalanine significantly increased the number of Ki67 positive cells in the jejunal crypts (P< 0.01). In vitro, β-alanine increased the organoid budding rates (P= 0.001) and the budding height of the crypt significantly (P= 0.004). In conclusion, β-alanine can improve intestinal morphology and barrier function, reduce inflammatory responses and alleviate the adverse effects of weaning stress on piglet intestinal health.