Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms including deficits in social interaction, repetitive and stereotyped behaviors, along with higher levels of anxiety and cognitive impairments. Previous studies demonstrate pronounced reduced density of calretinin (CR)-expressing GABAergic interneurons in both ASD patients and animal models. The object of the current study was to determine the role of CR in ASD-relevant behavioral aberrations. Herein, the mRNA and protein levels of CR in the prefrontal cortex (PFC) of mouse model of ASD based on prenatal exposure to valproic acid (VPA) were determined by qRT-PCR and Western blot analysis, respectively. Moreover, the behavioral abnormalities in naive mice with CR deficiency mediated by recombinant adeno-associated virus (rAAV) were evaluated in a comprehensive testing battery including social interaction, marble burying, self-grooming, open-field, elevated plus maze and novel object recognition tests. Furthermore, the action potential changes caused by CR deficiency were examined in neurons within the PFC in naive mouse. The results show that the mRNA and protein levels of PFC CR of VPA-induced mouse ASD model were reduced. Concomitantly, mice with CR knockdown displayed ASD-like behavioral aberrations, such as social impairments, elevated stereotypes, anxiety and memory defects. Intriguingly, patch-clamp recordings revealed that CR knockdown provoked decreased neuronal excitability by increasing action potential discharge frequencies together with decreased action potential threshold and rheobase. Our findings support a notion that CR knockdown might contribute to ASD-like phenotypes, with the pathogenesis most likely stemming from increased neuronal excitability.