Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3-deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3-deficient mice. These mice also exhibited a reduced rectification of AMPA receptor-mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3-deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD.