Fragile X syndrome (FXS) is the most common inherited intellectual disability, caused by a lack of the fragile X mental retardation protein (FMRP). Individuals with neurodevelopmental disorders frequently experience gastrointestinal problems that are primarily linked to gut microbial dysbiosis, inflammation, and increased intestinal permeability. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) are non-pharmacological agents that exert potential therapeutic effects against neurological disorders. However, it is unclear whether omega-3 PUFAs improve autistic behaviors in fragile X syndrome (FXS) by altering the gut microbial composition. Here, we describe gastrointestinal problems in Fmr1 knockout (KO) mice. FMRP deficiency causes intestinal homeostasis dysfunction in mice. Fish oil (FO) as a source of omega-3 PUFAs reduces intestinal inflammation but increases the mRNA and protein levels of TJP3 in the colon of juvenile Fmr1 KO mice. Fecal microbiota transplantation from FO-fed Fmr1 KO mice increased the gut abundance of Akkermansia and Gordonibacter in recipient Fmr1 KO mice and improved gut homeostasis and autistic behaviors. Our findings demonstrate that omega-3 PUFAs improve autistic behaviors and gut homeostasis in FMRP-deficient mice by suppressing gut microbiota dysbiosis, thereby presenting a novel therapeutic approach for juvenile FXS treatment.