Objective To investigate the expression of c-Jun N-terminal kinase (JNK) proteins in children with autism spectrum disorder (ASD) and the role of JNK in the pathogenesis of ASD.Methods A total of 30 children with ASD who were diagnosed in Hunan Children's Hospital from June 2016 to June 2017 were enrolled as ASD group, and 30 healthy children were enrolled as control group. Western blot was used to measure the expression levels of total JNK (T-JNK) and phosphorylated JNK (p-JNK) in peripheral blood mononuclear lymphocytes (PBMCs), Childhood Autism Rating Scale (CARS) was used to evaluate disease severity, and a correlation analysis was performed for JNK activation level and CARS score. Primary neuronal cells were transfected with recombinant adenovirus vector and were divided into JNK over-expression group and control group according to the intervention measures. The expression levels of T-JNK, p-JNK, phosphorylated ATF-2 (p-ATF-2), phosphorylated c-Jun (p-c-Jun), vesicular glutamate transporter (VGLUT), and vesicle-type GABA transporter (VGAT) were measured for both groups.Results There was no significant difference in the expression level of T-JNK in PBMCs between the ASD group and the control group (P>0.05), but the ASD group had significantly higher expression level of p-JNK and p-JNK/T-JNK ratio than the control group (P<0.05). The univariate logistic regression analysis was used to investigate the correlation between peripheral blood p-JNK/T-JNK ratio and CARS score, and the results showed that there was no correlation between JNK activation level and disease severity (r=0.03, P>0.05). After the primary neuron cells were transfected with adenovirus vector, the JNK over-expression group had significantly higher expression levels of T-JNK, p-JNK, p-c-Jun, and p-ATF-2 and a significantly lower expression level of VGAT than the control group (P<0.05), while there was no significant difference in the expression level of VGLUT between the two groups (P>0.05).Conclusions The JNK signaling pathway may be involved in the pathogenesis of ASD, but it is not associated with disease severity. Upregulation of JNK expression levels can activate the transcription factors ATF-2 and c-Jun and downregulate the expression of VGAT in synaptic vesicle transporters.