Repeated exposure to morphine leads to the addiction, which influences its clinical application seriously. The glutamatergic projection from prefrontal cortex (PFC) to the nucleus accumbens (NAc) plays an important role in rewarding effects. It is still unknown whether morphine exposure changes PFC-NAc synaptic transmission. To address this question, in vivo field excitatory postsynaptic potentials (fEPSPs) induced by electric stimulating PFC-NAc projection fibers were recorded to evaluate the effect of acute morphine exposure (10 mg/kg, s.c.) on glutamatergic synaptic transmission in NAc shell of repeated saline/morphine pretreated rats. It was showed that acute morphine exposure enhanced fEPSP amplitude and reduced paired-pulse ratio (PPR) in saline pretreated rats, which could be reversed by following naloxone injection (1 mg/kg, i.p.), an opiate receptor antagonist. However, repeated morphine pretreatment significantly inhibited both the enhancement of fEPSP amplitude and reduction of PPR induced by acute morphine exposure. Those results indicate that the initial morphine exposure enhances PFC-NAc synaptic transmission by pre-synaptic mechanisms, whereas morphine pretreatment occludes this effect.