High broodiness in Muscovy ducks impedes animal husbandry growth. The interaction between endocrine hormones and gut microbiota has been proven to play a crucial role in reproductive performance, and whether it can regulate the broody behavior of Muscovy ducks requires further research. Nine laying ducks (Laying group) and nine broody ducks (Broodiness group) were selected. Corresponding serum, ileum, and cecum chyme were collected for further research. The results showed that, compared to the laying group, the serum concentration of prolactin decreased, while the levels of Mullerian inhibiting substance, follicle-stimulating hormone, and follistatin increased in the broodiness group (P < 0.05). 16S rDNA sequencing showed that, the broodiness group exhibited lower abundance levels of Rothia, Streptococcus, and Lactobacillus, whereas the abundance of Turicibacter, Aliicoccus, and Facklamia was higher in the ileum compared to the laying group (P < 0.05). In the cecum, the broodiness group exhibits a significant reduction in the abundance of Butyricicoccus and unclassified_f_Rikenellaceae, while the abundance of Christensenellaceae_R-7_group, Ruminococcus_torques_group, Parabacteroides, norank_f_Oscillospiraceae, Cloacibacillus, Sellimonas, Shuttleworthia, norank_f_UCG-010, unclassified_f_Lachnospiraceae, Oscillospira, Synergistes, Family_XIII_AD3011_group and Eubacterium_nodatum_group is higher compared to the laying group. A Spearman correlation analysis reveals that both in the ileum and cecum, serum hormones exhibit significant correlations with the top 20 abundant intestinal microbial genera. Among these, serum follistatin has most entries of significant correlations with the detected microbial genera (P < 0.05). In conclusion, the broody behavior of Muscovy ducks can be modulated by the interaction between hormones and gut microbiota. Notably, the relationship between Follistatin and the composition of gut microbiota, specifically Firmicutes, is the most prominent.