It has been reported that dietary administration of Bacillus subtilisKC1 is effective in alleviating lung injury induced by Mycoplasma gallisepticum(MG) infection in chickens. However, the underlying molecular mechanism of B. subtilisKC1 against MG infection is still unclear. The purpose of this study was to determine whether B. subtilisKC1 could alleviate MG infection-induced lung injury in chickens by regulating their gut microbiota. The results of this study indicate that B. subtilisKC1 supplementation has the potential to alleviate MG infection-induced lung injury as reflected by reduced MG colonization, reduced pathologic changes, and decreased production of pro-inflammatory cytokines. In addition, B. subtilisKC1 supplementation was partially effective in alleviating the gut microbiota disorder caused by MG infection. Importantly, B. subtilisKC1 enriched the beneficial Bifidobacterium animalisin gut and thus reversed indole metabolic dysfunction caused by MG infection. B. subtilisKC1 supplementation increased levels of indole, which enhanced aryl hydrocarbon receptor activation, improving barrier function and alleviating lung inflammation caused by MG. Overall, this study indicates that B. subtilisKC1 has a “gut-lung axis” mechanism that can reduce the severity of MG infection by enriching intestinal B. animalisand regulating indole metabolism.