This study investigated the effect of Lactobacillus plantarum strain 299v on gut health in suckling piglets. Sixty newborn piglets were assigned to control and probiotic treatments, with three litters per treatment (ten piglets/litter). From days 1 to 20 of life, piglets were orally administered a placebo of 0.1% peptone or 1.0 ×  1010  CFU L. plantarum 299v daily. Six piglets per treatment were sacrificed on day 20, and intestinal tissues (including duodenum, jejunum, ileum and colon) and the intestinal contents from colon segments were collected. The results demonstrated that piglets treated with L. plantarum 299v had a lower diarrhoea incidence than the controls. L. plantarum 299v administration significantly increased the ratio of the villus height to the crypt depth in the jejunum and ileum, as well as the mRNA expression of jejunal occludin and ileal zonula occludens 1 (ZO-1). The L. plantarum treatment also increased the mRNA abundance of porcine β-defensin 2 (pBD2) and pBD3 in the jejunum and ileum and of toll-like receptors (TLRs), such as TLR2, TLR4, TLR6 and TLR9 in the ileum, and significantly upregulated the mRNA abundances of ileal pBD1 and colonic TLR4. Additionally, the L. plantarum 299v treatment significantly changed the structure of the colonic microbiota, as evidenced by the obvious increases in the relative abundances of the phyla Firmicutes and Actinobacteria and of the genus Lactobacillus. Our findings indicate that L. plantarum 299v facilitates the gut health of suckling piglets, probably by improving the intestinal morphology and intestinal barrier function and by modifying the structure of the gut microbiota.