Two studies were performed to assess the efficacy of Lactobacillus plantarum B1 in prevention of pathogenic Escherichia coli K88 gastrointestinal infection in broilers. In an in vitro study, L. plantarum B1 showed resistance to acid and bile and inhibited the growth of E. coli K88. Additionally, L. plantarum B1 exhibited high ability to adhere to broiler embryo ileal epithelium. In an animal trial, 240 broilers at 1 d of age were randomly assigned to one of 4 treatment arms: negative control (NC) broilers fed a basal diet and not challenged; positive control (PC) broilers fed a basal diet and challenged with E. coli K88; L. plantarum (LP) treatment broilers fed a basal diet containing 2 × 109 cfu/kg L. plantarum B1 and challenged with E. coli K88; and antibiotic treatment (Anti) broilers fed a basal diet supplemented with colistin sulfate (20 mg/kg) and challenged with E. coli K88. Broilers fed L. plantarum B1 had greater (P ≤ 0.05) BW than those in the PC treatment on d 14 and 28. Dietary L. plantarum B1 decreased (P < 0.05) E. coli counts in the cecal contents on d 10 and 14, and increased (P < 0.05) cecal lactic acid bacteria (LAB) on d 8, 10, 14, and 28 compared with the PC treatment. Dietary supplementation of L. plantarum B1 increased (P < 0.05) the ileal mucosal secretory IgA concentration and reduced (P < 0.05) IL-2, IL-4, IFN-γ, and tumor necrosis factor-α levels in the ileum. Overall, these results suggest dietary supplementation of L. plantarum B1 promotes growth performance, lowers cecal E. coli counts, and increases the population of cecal LAB, as well as improves intestinal mucosal immunity in E. coli K88-challenged broilers.