Rationale <p>Autism spectrum disorder (ASD) is characterized by cognitive deficits, repetitive behaviors, and social impairments. The SH3 and multiple ankyrin repeat domains protein 3B-deficient (Shank3B^-/-) mouse model displays ASD-related phenotypes. While rosmarinic acid (RosA) is known for its neuroprotective properties, its role in ASD remains unclear.</p> Objective <p>This study aimed to investigate the therapeutic effects and potential molecular mechanisms of RosA in alleviating behavioral dysfunction in Shank3B^−/− mice. We assessed core ASD-like behavioral indices, performed bioinformatics predictions, and validated the results through molecular biology experiments.</p> Methods <p>Social deficits were evaluated using the three-chamber social test and the male-male social interaction test. Repetitive behaviors were assessed through the self-grooming and marble-burying tests. Cognitive and memory functions were measured using novel object recognition, the Y-maze, and nesting behavior tests. The open field test was employed to evaluate motor functions and exploratory activities. High-throughput RNA sequencing (RNA-seq) was used to identify key genes in the medial prefrontal cortex (mPFC) of the different groups of mice. Neurotransmitter levels of acetylcholine (ACh) and γ-aminobutyric acid (GABA) were analyzed via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively. Additionally, synaptic function and plasticity in the mPFC were assessed by measuring Postsynaptic Density Protein 95 (PSD95) expression and the activation of the p-CREB/BDNF signaling pathway.</p> Results <p>RosA significantly improved repetitive behaviors, as well as cognitive and memory abilities, in Shank3B^-/- mice. It also enhanced motor functions and exploratory activities. However, RosA did not show significant therapeutic effects on social deficits. RNA-seq analysis revealed that RosA notably regulated synaptic proteins. Molecular biology experiments indicated that RosA upregulated PSD95 expression and activated the p-CREB/BDNF signaling pathway in the mPFC, enhancing synaptic plasticity. RosA also increased ACh levels without affecting GABA, indicating a cholinergic mechanism. No significant effects were observed in wild-type (WT) mice, suggesting specificity to ASD-related deficits.</p> Conclusion <p>RosA alleviates cognitive deficits and repetitive behaviors in Shank3B^-/- mice through CREB/BDNF-mediated synaptic and cholinergic regulation in the mPFC. However, its lack of effect on social deficits suggests distinct mechanisms underlying ASD symptoms. These findings highlight the potential of RosA as a targeted ASD therapy.</p> Graphical Abstract <p></p>