Abstract: Objective: To identify potential imaging biomarkers for the early diagnosis of autism spectrum disorder (ASD) and further reveal the possible neuroimaging mechanisms underlying the severity of ASD symptoms, this study investigated brain abnormalities and resting-state functional connectivity (RSFC) in preschool-aged children with ASD compared to typically developing (TD) children using magnetic resonance imaging (MRI). The study also evaluated the correlation between functional connectivity and Childhood Autism Rating Scale (CARS) scores. Methods: This prospective study involved 20 preschool-aged children with ASD (aged 3-6 years) and 20 age-matched TD children, who underwent structural and resting-state functional MRI (rs-fMRI) scans. First, based on the structural MRI data, voxel-based morphometry (VBM) was used to identify the differences in gray matter volume (GMV) between the two groups. Based on the VBM results, the brain region with the most significant GMV difference was used as the seed region of interest for the RSFC analysis. Results from both analyses (Rs-fMRI and seed-based functional connectivity analysis) were used to calculate the RSFC values between significant brain regions and the whole brain. Subsequently, the correlation between RSFC values and CARS scores was assessed. Results: Whole-brain VBM analysis revealed a significantly lower GMV in the right insula in the ASD group than in the TD group. The RSFC results showed that the weaker the FC values between the right insula and the left anterior cingulate cortex, the higher the corresponding clinical ASD symptom score, indicating a negative correlation with the CARS scores (r=−0.758). Conclusion: A lower GMV in the right insula may serve as an imaging biomarker for preschool-aged children with ASD. The difference in functional connectivity between the right insula and left anterior cingulate cortex may further reveal the neural underpinnings associated with ASD symptoms.