Abstract Dietary probiotics were used in the animal feeds because of the beneficial effect of improving gut health and immune responses as one of the potential alternatives to antibiotics. This study was conducted to evaluate the effect of dietary supplementation of Lactobacillus rhamnosus (ILDONG Bioscience Co. Ltd., Pyeongtaek, Republic of Korea) on blood profiles, small intestinal morphology, immune responses, and gene expression of tight junction proteins and inflammatory cytokines in ileum of weaned pigs. A total of 40 weaned pigs [6.21 ± 0.55 kg of initial body weight (BW); 28-day old] were allotted to 2 dietary treatments (4 pigs/pen; 5 replicates/treatment) in a randomized complete block design (block: initial BW) and fed for 4 weeks. Dietary treatments were 1) a commercial basal nursery diet (CON), and 2) CON + 0.02% Lactobacillus rhamnosus IDCC 3201 (PRO). The blood samples were collected from randomly selected 1 pig per pen on day 1, 7, 14, and 28 for blood profiles and immune responses. On the last day of experiment, the pigs were euthanized to collect duodenal, jejunal, and ileal segments for intestinal morphology and gene expression analysis. Intestinal morphology was measured villus height, width, area, crypt depth, villus height to crypt depth ratio, and number of goblet cells by hematoxylin and eosin staining method. The blood profiles were measured by using an automated hematology analyzer. The serum samples were used to measure concentrations of cytokines and cortisol by using porcine immunoassay ELISA kits. The gene expression of tight junction proteins and cytokines were measured by qRT-PCR. Data were analyzed by PROC GLM procedure of SAS. Experimental unit was a pen. Statistical model for blood profiles, immune responses, and intestinal morphology included dietary treatment as a main effect and initial BW as a covariate. The t-test was used for ileal gene expression. Pigs fed PRO had lower (P < 0.05) platelet level, crypt depth in duodenum and ileum, serum tumor necrosis factor-α (TNF-α), and interleukin-6 than those fed CON. The pigs fed PRO downregulated (P < 0.05) TNF-α and interleukin-8 genes compared with those fed CON, but upregulated (P < 0.05) claudin-1 and claudin-2 genes. In conclusion, probiotic Lactobacillus rhamnosus supplementation in nursery diet improved gut health through modulating blood profiles and immune responses, enhancing intestinal morphology, and regulating tight junction protein genes expression in ileum of weaned pigs.