The anticancer drug oxaliplatin is associated with peripheral neuropathy as a side effect accompanied by mechanical and cold allodynia. Although the superficial layer of the spinal cord dorsal horn is known to receive information primarily from peripheral pain nerves, to our knowledge, no in vivo electrophysiological analyses have been conducted to determine whether oxaliplatin administration increases the excitability of superficial layer neurons. Therefore, in vivo extracellular recordings were performed to measure action potentials in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose (6 mg/kg) of oxaliplatin. Action potentials were produced by mechanical stimulation with von Frey filaments to the hindlimb receptive fields. The results revealed that the firing frequency of action potentials increased relative to the intensity of mechanical stimulation, and that both deep and superficial layer neurons in the spinal cord dorsal horn increased significantly in oxaliplatin-treated compared with vehicle-treated rats, especially in the superficial layer. Several superficial layer neurons showed spontaneous firing that was not seen in vehicle-treated rats. In addition, a clear increase was seen in the firing frequency of neurons in the superficial layer of oxaliplatin-treated rats in response to a cold stimulus (here, the addition of acetone to the hindlimb receptive field). This study suggests that the superficial spinal cord dorsal horn strongly reflects the pain pathophysiology in peripheral neuropathy induced by oxaliplatin administration, and that the superficial layer neurons are useful for in vivo electrophysiological analysis using this pathological model.