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Background:Probiosis is considered a potential strategy to reduce antibiotics use and prevent post-weaning diarrhea (PWD). This study investigated the effect of Bacillus amyloliquefaciens DSM25840 or Bacillus subtilis DSM25841 supplementation on growth, health, immunity, intestinal functionality and microbial profile of post-weaning pigs after enterotoxigenic E. coli (ETEC) F4 challenge. Methods: Sixty-four post-weaning piglets (7748 g ± 643 g) were randomly allocated to four groups:control basal diet (CO);CO+1.28 × 106 CFU/g of B. amyloliquefaciens (BAA);CO+1.28 × 106 CFU/g feed of B. subtilis (BAS);CO+1 g colistin/kg of feed (AB). At day (d) 7, animals were challenged with 105 CFU/mL of ETEC F4ac O149 and then followed for fecal score and performance until d 21. Blood was collected at d 6, d 12 and d 21 for immunoglobulins, at d 8 for acute phase proteins, at d 8 and d 21 for metabolomics analysis. Jejunum was sampled for morphometry, quantification of apoptosis, cell proliferation, neutral and acid mucine and IgA secretory cells, and microarray analysis at d 21. Jejunum and cecum contents were collected for microbiota at d 21. Results:AB and BAS reduced the fecal score impairment compared to CO (P<0.05) at d 14. Body weight (BW), average daily weight gain (ADWG), average daily feed intake (ADFI) and gain to feed ratio (G:F) did not differ between Bacillus groups and CO. AB improved BW at d 7, d 14 and d 21, ADWG ADFI and G:F from d 0 to d 7 (P<0.05). At d 8, CO had higher plasma arginine, lysine, ithine, glycine, serine and threonine than other groups, and higher haptoglobin than AB (P<0.05). At d 21, CO had lower blood glycine, glutamine and IgA than BAS. Morphology, cells apoptosis and mucins did not differ. BAS and AB increased the villus mitotic index. Transcriptome profile of BAS and AB were more similar than CO. Gene sets related to adaptive immune response were enriched in BAA, BAS and AB. CO had enriched gene set for nuclear structure and RNA processing. CO had a trend of higher Enterobacteriaceae in cecum than the other groups (P=0.06). Conclusion:Bacillus subtilis DSM25841 treatment may reduce ETEC F4ac infection in weaned piglets, decreasing diarrhea and influencing mucosal transcriptomic profile.