Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder, characterized by impaired social interaction, repetitive behavior and restricted interests. Although the molecular etiology of ASD remains largely unknown, recent studies have suggested that de novo mutations are significantly involved in the risk of ASD. We and others recently identified spontaneous de novo mutations in PKD2, a protein kinase D family member, in sporadic ASD cases. However, the biological significance of the de novo PKD2 mutations and the role of PKD2 in brain development remain unclear. Here, we performed functional analysis of PKD2 in cortical neuron development using in utero electroporation. PKD2 is highly expressed in cortical neural stem cells in the developing cortex and regulates cortical neuron development, including the neuronal differentiation of neural stem cells and migration of newborn neurons. Importantly, we determined that the ASD-associated de novo mutations impair the kinase activity of PKD2, suggesting that the de novo PKD2 mutations can be a risk factor for the disease by loss of function of PKD2. Our current findings provide novel insight into the molecular and cellular pathogenesis of ASD.