Substance P is one of the major neuropeptides released by striatal neurons; however, its function in the striatum remains unclear. In this study, we found substance P triggers spontaneous neurotransmitter release and rapid synaptic vesicle exocytosis in cultured striatal neurons, as substance P knockdown in these neurons impaired spontaneous neurotransmitter release and calcium-dependent rapid synaptic neurotransmission. Furthermore, treatment with exogenous substance P completely rescued the synaptic dysfunction phenotype in striatal neurons lacking this neuropeptide. On the other hand, substance P knockdown had no effect on the size of the readily releasable pool of synaptic vesicles, but decreased the probability of presynaptic release of synaptic vesicles in cultured striatal neurons. Treatment with CP96345, a NK1 receptor antagonist, also resulted in synaptic defects in cultured striatal neurons. In summary, we propose substance P is critical for synaptic transmission in striatal neurons. (C) 2019 Elsevier Inc. All rights reserved.