<p>Dysregulation of microRNAs (miRNAs) has emerged as a critical factor in the pathophysiology of Autism spectrum disorder (ASD), particularly in the disruption of gene networks involved in neurodevelopment. AST-001, an L-serine derivative, has shown potential to improve ASD-like behaviors by modulating dopaminergic signaling, but its underlying molecular mechanisms remain unclear. This study aimed to explore whether AST-001 exerts therapeutic effects by regulating ASD-related gene expression through miRNA modulation. Male offspring exposed to VPA were treated with AST-001 for two weeks. Midbrain samples were collected from control, VPA, and AST-001-treated VPA groups. qPCR and small RNA sequencing (small RNA-seq) were performed to profile gene and miRNA expression. The regulatory relationships between miRNAs and ASD-related genes were evaluated using dual-luciferase assays and qPCR. Among seven ASD-related genes examined, DLG4, PAX5, and SNAP23 were significantly upregulated in the VPA model and downregulated following AST-001 treatment. Small RNA-seq and qPCR identified four miRNAs (mmu-miR-378c, mmu-miR-483-5p, mmu-miR-3085-3p, and mmu-miR-128-3p) that were downregulated in the VPA group and restored toward Control levels after AST-001 treatment. Gene ontology analysis showed that these miRNAs are involved in synaptic signaling and neurodevelopmental processes associated with ASD. Their predicted binding to the 3′ UTRs of the ASD-related genes was further analyzed and characterized. These findings suggest a potential mechanism by which AST-001 modulates miRNA-mediated regulation of genes involved in synaptic function and brain development, including <i>DLG4</i>, <i>PAX5</i>, and <i>SNAP23</i>. This post-transcriptional modulation highlights a plausible pathway to help rebalance disrupted dopaminergic and synaptic signaling networks, offering early mechanistic insights into the therapeutic potential of AST-001.</p>