AbstractAimsThe purpose of this study was to investigate the effects of Bacillus subtilis supplementation on the health of weaned piglets and whether B. subtilis supplementation can reduce the damage of piglets induced by ETEC K88.Methods and resultsThe experiment was designed with a 2 × 2 factorial arrangement, comprising the control group, B. subtilis (PRO) group, Escherichia coli K88 (ETEC) group, and B. subtilis + ETEC (PRO + ETEC) group. Regardless of the presence of ETEC, the addition of PRO increased the piglets’ final body weight, average daily gain, and daily feed intake. Additionally, PRO primarily achieves a reduction in heat-stable enterotoxin (ST) levels, suppresses the expression of NF-κB, TLR4, and MyD88 mRNA in the jejunum and ileum, lowers pro-inflammatory factors in the blood and small intestine, enhances the expression of tight junction proteins in the small intestine, improves the composition of the colonic microbiota, increases colonic short-chain fatty acid contents, thereby alleviating diarrhea and mitigating bodily damage caused by ETEC K88 infection.ConclusionThe addition of B. subtilis MZ-01 alleviated ETEC K88-induced piglet diarrhea by reducing ST levels, decreasing pro-inflammatory factors in the blood and intestine, and enhancing the intestinal barrier and tight junction proteins.