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AbstractCurrent experiment was performed in order to investigate the effects of Bacillus subtilis-based probiotic supplementation and fermented soybean meal (FSBM) on gut health, immune response to Newcastle disease virus (NDV), dry matter digestibility and growth performance in broiler chickens. Three hundred 1-day-old male broiler chicks (Ross 308) were allocated to five dietary treatments using a completely randomised design. Each treatment was replicated six times with 10 birds/replicate. The dietary treatments were (1) control group [basal diet (BD), without probiotic and FSBM], (2) BD + 0.02% probiotic (0.8 × 105CFU of spores/gr feed), (3) BD + 0.04% probiotic (1.6 × 106CFU of spores/gr feed), (4) 10% replacement of soybean meal (SBM) with FSBM in BD and (5) 20% replacement of SBM with FSBM in BD. Soybean fermentation was carried out under solid fermentation condition by B. subtilisspore. FSBM replacement at 20% improved weight gain and feed conversion ratio compared with the control group (p < 0.05). FSBM replacement at the level of 20% led to increase in villi height (p < 0.05). Villus height in the ileum was increased by 0.04% probiotic supplementation. FSBM replacement improved chicks’ immune response against NDV vaccine on day 42 (p < 0.05), but the effect was not significant for probiotic supplementation. Coliforms count in ileum was reduced by probiotic and FSBM supplementation (p < 0.05). Dry matter digestibility was improved by probiotic supplementation at 0.04%. According to the results of the current study, replacing SBM with FSBM at 20% and dietary supplementation of B. subtilisspores improved growth performance, intestinal health, immune system and dry matter digestibility of broilers.HighlightsFermentation is one of the most effective methods which can be used to decrease the feed anti-nutritional factors and to improve feed nutritional properties.Both the direct fed microbe via Bacillus subtilisspores supplementation and replacement of fermented soybean meal (FSBM) instead of soybean meal improved the chicks’ performance which mostly can be related to the beneficial effects of B. subtilisand FSBM on the gut health.