Vitamin B6 (VB6) exhibits therapeutic effects towards autism spectrum disorder (ASD), but its specific mechanism is poorly understood. Rat dams were treated with VB6standard, VB6deficiency, or VB6supplementary diet, and the same treatment was provided to their offspring, with their body weights monitored. Three-chambered social test and open field test were employed to evaluate the effect of VB6on autism-like behaviors. Gamma-aminobutyric acid (GABA) generation and synaptic inhibition of neurons in the hippocampus of rat were detected via immunofluorescence staining, followed by the measurement of GABA concentration through high-performance liquid chromatography (HPLC). The role of VB6in the autophagy and apoptosis of cells was determined via Western blot and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). In order to conduct rescue experiments, the inhibition of mammalian target of rapamycin (mTOR) or the activation of GABA was achieved by drug administration to the offspring rats with VB6deficiency. As a result, no evident difference in weight was observed in the offspring with varied VB6treatments. VB6deficiency impaired social interaction; aggravated self-grooming and bowel frequency; decreased GABA concentration, VIAAT, GAD67, vGAT expressions, and LC3 II/LC3 I ratio; increased p62 level and p-mTOR/mTOR ratio; and promoted cell apoptosis. Inhibition of mTOR reversed the effect of VB6deficiency on cell autophagy. GABA activation or mTOR inhibition offset the role of VB6deficiency in autism-like behaviors and hippocampal GABA expression. Collectively, VB6deficiency induces autism-like behaviors in rats by regulating mTOR-mediated autophagy in the hippocampus.