It has been shown that activation of the glutamatergic transmission in the prelimbic region of the mPFC (PL-PFC) evoked anxiety-like behavior in rodents. We previously reported that local perfusion of a selective agonist to delta-opioid receptor (DOP), KNT-127, attenuated the veratrine-induced elevation of extracellular glutamate in the PL-PFC and anxiety-like behavior in mice. These results suggested the possibility that KNT-127 suppresses glutamate release from presynaptic site in the PL-PFC. To confirm this, we performed whole-cell patch-clamp recording from pyramidal neurons in the PL-PFC, and examined the spontaneous and electrically-evoked excitatory post-synaptic currents (EPSC)s. We found that bath application of KNT-127 significantly suppressed frequency, but not amplitude of spontaneous and miniature EPSCs in a DOP-dependent manner. Also, KNT-127 increased paired-pulse ratios in the PL-PFC pyramidal neurons tested. Further, we analysed the firing properties of pyramidal neurons in the PL-PFC, and found that KNT-127 treatment significantly reduced the number of action potentials and rheobase. These results suggested that KNT-127 not only suppresses glutamatergic synaptic transmission by inhibiting glutamate release from presynaptic site, but also reduces cell excitability via DOP in the mouse PL-PFC.