Currently, opioid-based drugs are the most effective pain relievers that are widely used in the treatment of pain. However, the analgesic efficacy of opioids is significantly limited by the development of tolerance after repeated opioid administration. Glutamate receptors have been reported to critically participate in the development and maintenance of opioid tolerance, but the underlying mechanisms remain unclear. Using whole-cell voltage-clamp recordings in brainstem slices, the present study investigated chronic morphine-induced adaptations in glutamatergic synaptic transmission in neurons of the nucleus raphe magnus (NRM), a key supraspinal relay for pain modulation and opioid analgesia. Chronic morphine significantly increased glutamate synaptic transmission exclusively in one class of NRM cells that contains mu-opioid receptors in a morphine-tolerant state. The adenylyl cyclase activator forskolin and the cAMP analog 8-bromo-cAMP mimicked the chronic morphine effect in control neurons and their potency in enhancing the glutamate synaptic current was significantly increased in neurons from morphine-tolerant rats. MDL12330a, an adenylyl cyclase inhibitor, and H89, a protein kinase A (PKA) inhibitor, reversed the increase in glutamate synaptic transmission induced by chronic morphine. In addition, PMA, a phorbol ester activator of protein kinase C (PKC), also showed an increased potency in enhancing the glutamate synaptic current in these morphine-tolerant cells. The PKC inhibitor GF109203X attenuated the chronic morphine effect. Taken together, these results suggest that chronic morphine increases presynaptic glutamate release in mu receptor-containing NRM neurons in a morphine-tolerant state, and that the increased glutamate synaptic transmission appears to involve an upregulation of both the cAMP/PKA pathway and the PKC pathway. This glutamate-mediated activation of these NRM neurons that are thought to facilitate spinal pain transmission may contribute to the reduced opioid analgesia during opioid tolerance.