Bacillus is widely used in the livestock industry. This study was designed to evaluate the effects of probiotic Bacillus amyloliquefaciens SC06 (Ba), originally isolated from soil, in piglets diet as an alternative to antibiotics (aureomycin), mainly on intestinal epithelial barrier and immune function. Ninety piglets were divided into three groups: G1 (containing 150 mg/kg aureomycin in the diet); G2 (containing 75 mg/kg aureomycin and 1 x 10(8) cfu/kg Ba in the diet); G3 (containing 2x 10(8) cfu/kg Ba in the diet without any antibiotics). The results showed that, compared with the antibiotic group (G1), villus length, crypt depth and villus length/crypt depth ratio of intestine significantly increased in the G2 and G3 groups. In addition, intestinal villi morphology, goblet-cell number, mitochondria structure and tight junction proteins of intestinal epithelial cells in G2 and G3 were better than in G1. The relative gene expression of intestinal mucosal defensin-1, claudin3, claudin4, and human mucin-1 in G3 was significantly lower, while the expression of villin was significantly higher than in the antibiotic group. Probiotic Ba could significantly decrease serum interferon (IFN)-IFN-gamma, interleukin (IL)-1 beta, and IL-4 levels, whereas increase tumour necrosis factor (TNF)-alpha and IL-6 secretion. Ba could also significantly decrease cytokines TNF-alpha, IFN-gamma, IL-1 beta, and IL-4 level in liver, whereas it significantly increased IFN-alpha. Furthermore, replacing antibiotics with Ba also significantly down-regulated gene expression of TNF and IL-1 alpha in intestinal mucosa, but up-regulated IL-6 and IL-8 transcription. Dietary addition of Ba could significantly reduce the gene expression of nuclear factor kappa beta (NF kappa B)-p50 and Toll-like receptor (TLR)6, while there was no significant difference for that of myeloid differentiation primary response 88, TNF receptor-associated factor-6, nucleotide-binding oligomerisation domain containing protein 1, TLR2, TLR4, and TLR9. Taken together, our findings demonstrated that probiotic Ba could increase the intestinal epithelial cell barrier and immune function by improving intestinal mucosa structure, tight junctions and by activating the TLRs signalling pathway.