The development of new strategies for the treatment of acute pain requires the identification of novel nonopioid receptor targets. This study explored whether δ-subunit-containing GABA(A)Rs (δGABA(A)Rs) in neurons of the spinal cord dorsal horn generate a tonic inhibitory conductance in vitro and whether δGABA(A)R activity regulates acute nociception. Whole-cell recordings revealed that δGABA(A)Rs generate a tonic inhibitory conductance in cultured spinal neurons and lamina II neurons in spinal cord slices. Increasing δGABA(A)R function by applying the δGABA(A)R-preferring agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP) increased the tonic current and inhibited neuronal excitability in spinal neurons from wild-type (WT) but not δ subunit null-mutant (Gabrd(-/-)) mice. In behavioral studies, baseline δGABA(A)R activity did not regulate acute nociception; however, THIP administered intraperitoneally or intrathecally attenuated acute nociception in WT but not Gabrd(-/-) mice. In the formalin nociception assay, the phase 1 response was similar for WT and Gabrd(-/-) mice. In contrast, the phase 2 response, which models central sensitization, was greater in Gabrd(-/-) mice than WT. THIP administered intraperitoneally or intrathecally inhibited phase 1 responses of WT but not Gabrd(-/-) mice and had no effect on phase 2 responses of WT mice. Surprisingly, THIP reduced the enhanced phase 2 response in Gabrd(-/-) mice. Together, these results suggest that δGABA(A)Rs in spinal neurons play a major physiological and pharmacological role in the regulation of acute nociception and central sensitization. Spinal δ-subunit-containing GABA(A) receptors were identified with electrophysiological methods and behavioral models as novel targets for the treatment of acute pain.