To investigate the effects of early supplementation of starter pellets with alfalfa in pre-weaning period on the permeability, expressions of tight junction (TJ) proteins and cytokines of gastrointestinal tract during the pre- and post-weaning period, 6 of 66 male Hu lambs at the age of 10 d were slaughtered as control, and the other 60 lambs were randomly allocated to 2 dietary treatments: milk replacer and starter pellets without (STA) or with free-choice provision of chopped alfalfa (S-ALF). The animals were offered 300-g/d concentrate mixture and had free access to alfalfa after weaning at the end of wk 4 (age of 38 d). The plasma concentrations of D-lactic acid, lipopolysaccharides, IgA, IgM and IgG were measured by ELISA kit (RB). Ruminal epithelium and duodenal mucosa were subjected to Ussing chamber to measure the transepithelial electrical resistance (TER) and conductance. The mRNA expressions of TJ proteins and cytokines of gastrointestinal tract were determined with qRT-PCR. Results were analyzed as a completely randomized design. All data were analyzed by 2-way ANOVA using PROC GLM SAS (version 9.2; SAS Institute Inc., Cary, NC). Data of TER and conductance showed that alfalfa supplementation increased or decreased permeability of ruminal epithelium and duodenal mucosa numerically in pre- or post-weaning period, respectively, while the plasma concentrations and the morphological appearance of duodenum were not different between 2 treatments (P > 0.05). Compared with STA group, alfalfa supplementation increased (P = 0.057) the ruminal occludin expression, enhanced (P < 0.01) the duodenal expression of claudin-1, and numerically enhanced ileal expression of claudin-1 and claudin-4 during the pre-weaning period, and inhibited (P < 0.05) the weaning-caused compensatory increase in ruminal epithelial expression of claudin-4. The change in ruminal expression of TNF-α with weaning was similar to claudin-4. In summary, early supplementation of alfalfa to starter diet could maintain the normal function of the gastrointestinal barrier, and helped to release the stress from weaning and feed transition in growing lambs.