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The aim of present study was to evaluate the effect of Limosilactobacillus reuteri (L. reuteri) on the intestinal barrier function and mucosal immunity of piglets at different suckling phase. In this study, fifty-four neonatal piglets (Rongchang × Yorkshire) were orally administered with 1.0 × 1010 CFU L. reuteri I5007 (dissolved in 2 ml 0.1% peptone) or 2 ml 0.1% peptone by gavage every day. The experiment had lasted for 20 days from the birth. Six piglets from each group were slaughtered at 10- and 20-day-old, respectively. Intestinal samples were collected for the evaluation of target genes by real-time PCR and western blot. The results showed that L. reuteri I5007 significantly increased the average daily gain, decreased the diarrhoea incidence, and improved the intestinal morphology of piglets (p <.05). Oral administration of L. reuteri I5007 also elevated the mRNA and protein level of jejunal and ileal Zonula Occludens-1, Claudin-1, and Occludin to enhance the intestinal mucosal barrier function. Additionally, L. reuteri I5007 treatment modulated toll-like receptors (TLRs) and porcine host defense peptides (HDPs) expression, such as increased the mRNA levels of ileal TLR9 and PBD2 at 10-day-old, jejunal TLR9 and ileal PG1-5 at 20-day-old, the protein level of ileal TLR2 at 20-day-old, and influenced the protein and mRNA level of ileal TLR4. These findings indicated that oral administration of L. reuteri I5007 could improve the intestinal mucosal barrier and enhance the intestinal innate immunity of piglets during suckling period. L. reuteri increased the average daily gain and decreased the diarrhoea incidence of piglets. L. reuteri increased the expression of Zonula Occludens-1, Claudin-1, and Occludin in jejunal and ileal mucosal. L. reuteri influenced the expression of toll-like receptors and porcine host defense peptides. [ABSTRACT FROM AUTHOR]