Brain-behavior-gut-microbiome interaction, a bidirectional communication, was proposed as an important role in autism spectrum disorders (ASD). However, the correlation among gut microbiota, behavioral performance, and brain microstructure in ASD are remained unclear. We chose a VPA-exposed rat model which performed autistic behaviors to investigate their brain-behavior-gut interaction. Diffusion MRI, behavioral tests, and intestinal bacteria gene analysis were applied in this study. The findings implied that the altered brain microstructure and atypical distribution of the gut microbiota associate with the severity of the autistic behavior in ASD compared to the control group.