Background:Emerging evidencesindicate a close relationship between gut microbiotaand fatty liver disease.It has been suggested that gut microbiota modulation with probiotics improved fatty liver disease in rodents and human, yet it still remains unclear in poultry.Results: Ninety six one-day-old green-legged chicken were divided into control group (CON) and probiotic group (PB), respectively. Probiotics were administrated throughdrinking water for two weeks from 1-day-old. At 28 d of age,16 broilers selectedfromCON or PB group randomly, and receivedvehicle(15% ethanol) or CORT(4.0 mg/kg)treatmentdaily viasubcutaneous injection for a week to induce fatty liver. At the end of the experiment, broilers from 4 groups,control group(CON), corticosterone group (CORT), probiotic group (PB), PB plusCORT group(CORT&PB),were slaughtered for sampling and analysis.The results showed that probiotics administration significantly prevented CORT-induced body weight loss(P<0.05), but did not alleviate the reduction of immune organs weight caused by CORT. Compared to CON,broilers in CORT group exhibited a significant increase of triglyceride (TG) levelin both plasma and liver(P< 0.01), as well as severe hepatocytic steatosis and hepatocellular ballooning, and accompanied with the up-regulation of hepatic lipogenesis genes expression. However, probiotics supplementation markedly decreased the intrahepatic lipid accumulation and steatosis histological score, which was associated withthedown-regulation of sterol regulatory element-binding protein-1 (SREBP1) and acetyl-CoA carboxylase (ACC) mRNA (P< 0.05)as well as it protein (P= 0.06) expression.Cecal microbiota composition was determined by 16S rRNA high-throughput sequencing. Results showed that CORT treatment induced distinct gut microbiota alterations with a decrease of microbial diversity, and anincrease of proteobacteriaabundance (P<0.05). On the contrary, probiotic supplementation increased the beta diversity and increased community richness and diversity index(P> 0.05), as well as the abundance of Intestinimonas(P<0.05).Conclusion: Our results indicate that CORT treatment induced serious fatty liver disease and altered the gut microbiota composition in broilers, however,probiotics supplementation from post-hatching had a beneficial effect on alleviatingfatty liver disease through regulating lipogenic genes expression and increasing gut microbiota diversity andbeneficial bacteria abundanceimbalance.