Presynaptic ATP P2X receptors have been proposed to play a role in modulating glutamate release from the first sensory synapse in the spinal cord. Using spinal cord slice preparations and patch-clamp recordings from dorsal horn neurons in lamina V of the rat spinal cord, we showed that the activation of P2X receptors by α,β-methylene-ATP (αβm-ATP) resulted in a large increase in the frequency of spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs). The increases in mEPSC frequency by αβm-ATP were not blocked by the Ca2+ channel blocker, 30 μM La3+, but were abolished in a bath solution when Ca2+ was omitted. The increases in mEPSC frequency by αβm-ATP were blocked completely by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) at 10 μM. Furthermore, the EPSCs evoked by dorsal root stimulation were potentiated by αβm-ATP as well as by the ecto-ATPase inhibitor ARL67156 and were depressed in the presence of P2 receptor antagonists PPADS (10 μM) and suramin (5 μM). The effects of these compounds on the evoked EPSCs were associated with the changes in glutamate release probability of primary afferent central terminals. Our results indicate that αβm-ATP-sensitive P2X receptors play a significant role in modulating excitatory sensory synaptic transmission in the spinal cord, and the potential role of endogenous ATP is suggested.