Summary This study was conducted to investigate the effects of aflatoxin (AF), Clostridium perfringen (CP) and yeast cell walls (YCW) on the growth performance, intestinal lesion, mucosal barrier, and microflora population of broilers. In the 21-d study, 576 one-day-old broiler chicks were assigned to a 2×2×2 factorial arrangement of treatments with 6 replicated pens. Experimental factors included a) AFB 1 0 or 40Â μg/kg of diet; b) CP challenge or unchallenged; and c) YCW 0 or 500Â mg/kg. Pens of birds were challenged on CP contaminated litter from d 1. Intestines of 5 birds per pen were examined for gut parameters on d 21. The results showed that based on the diets without CP challenge, dietary AF decreased ( P<Â 0.05) FI and BWG of broilers, and the CP challenge depressed ( P<Â 0.05) BWG. With the double challenges of AF and CP, the YCW increased ( P<Â 0.05) FI. Dietary AF increased ( P<Â 0.05) lesion scores in jejunum and ileum; whereas CP increased ( P<Â 0.05) lesion scores in duodenum, jejunum and ileum, but YCW decreased ( P<Â 0.05) ileal lesion scores. Dietary CP up-regulated ( P<Â 0.05) mucin 2 expression and down-regulated ( P<Â 0.05) sIgA level, whereas YCW up-regulated ( P<Â 0.05) sIgA and pIgR. Additionally, the counts of Lactobacilli and Bifidobacteria were decreased ( P<Â 0.05) by CP, but increased ( P<Â 0.05) by YCW. It can be concluded that YCW is helpful in improving intestinal barrier and microbiota, and decreasing risks to aflatoxin or necrotic enteritis infections.