It has been indicated that prostaglandin E₂ (PGE₂) and the receptor for PGE₂ (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE₂ and EP₁ receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca²⁺]_i) assays and in situ hybridization. Experiments were conducted on Sprague–Dawley rats. In behavioral assays, withdrawal thresholds to mechanical stimuli were evaluated using von Frey filament. The effect of an intrathecally administered selective EP₁ antagonist, 6-[(2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5Z-hexenoic acid (ONO-8711), on the intrathecal PGE₂-induced hyperalgesia was examined. In [Ca²⁺]_i assays, we measured [Ca²⁺]_i in the dorsal horn of spinal cord slices and examined the effects of PGE₂ and ONO-8711 perfusion on the [Ca²⁺]_i changes. In situ hybridization using EP₁ digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L₄ and L₅ dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE₂ administration. Intrathecal administration of ONO-8711 attenuated the PGE₂-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE₂-induced [Ca²⁺]_i increment in laminae II–VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP₁ hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE₂ activates the EP₁ receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca²⁺]_i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE₂-induced nociceptive transmission.