Astrocytes play a key role in the maintenance of synaptic transmission by producing L-lactate via the astrocyte-neuron lactate shuttle (ANLS). Astrocyte activation in the spinal cord is involved in the expression of neuropathic pain. These reactive astrocytes are suggested to play an important role in the maintenance of neuropathic pain. We investigated the role of the ANLS in the spinal cord on hyperalgesia in neuropathic pain in mice. We also investigated the cellular mechanisms of spinal L-lactate-induced mechanical hyperalgesia. We revealed that the selective activation of spinal dorsal horn astrocytes causes mechanical hyperalgesia through the excessive L-lactate supply to neurons via monocarboxylate transporters (MCTs). We also found that L-lactate transported into neurons may produce mechanical hyperalgesia. In addition, application of L-lactate enhanced the excitatory neurotransmission evoked by mechanical stimulation in the dorsal horn of spinal cord. Moreover, intrathecal treatment with L-lactate also activates protein kinase A (PKA) signaling. These results suggest that the enhanced ANLS sensitizes the nociceptive transmission in the spinal cord through the activation of PKA signaling.