Autism spectrum disorder (ASD) is a neurodevelopmental disorder displaying the modification of complex human behaviors, characterized by social interaction impairments, stereotypical/repetitive activities and emotional dysregulation. In this study, fecal microbiota transplant (FMT) via gavage from autistic children donors to mice, led to the colonization of ASD-like microbiota and autistic behaviors compared to the offspring of pregnant females exposed to valproic acid (VPA). Such variations seemed to be tightly associated with increased populations of Tenericutes plus a notable reduction (p < 0.001) of Actinobacteria and Candidatus S. in the gastrointestinal region of FMT mice as compared to controls. Indeed altered behaviors of FMT mice was reported when evaluated in the different maze tests (light dark, novel object, three chamber tests, novel cage test). Contextually, FMT accounted for elevated expression levels of the pro-inflammatory factors IL-1β, IL-6, COX-1 and TNF-α in both brain and small intestine. Villous atrophy and inflammatory infiltration (Caspase 3 and Ki67) were increased in the small intestine of FMT and VPA mice compared to controls. Moreover, the observed FMT-dependent alterations were linked to a decrease in the methylation status. Overall, findings of the present study corroborate a key role of gut microbiota in ASD. However, further investigations are required before any possible manipulation of gut bacteria with appropriate diets or probiotics can be conducted in ASD individuals.