BACKGROUND AND PURPOSE The sigma‐1 (σ1) receptor is a ligand‐regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ1 receptor ligands used as pharmacological tools are unclear and the demonstration that σ1 receptor antagonists have efficacy in reversing central sensitization‐related pain sensitivity is still missing. EXPERIMENTAL APPROACH The pharmacological properties of a novel σ1 receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ1 receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ1 receptor occupancy were measured to substantiate behavioural data. KEY RESULTS Formalin‐induced nociception (both phases), capsaicin‐induced mechanical hypersensitivity and sciatic nerve injury‐induced mechanical and thermal hypersensitivity were dose‐dependently inhibited by systemic administration of S1RA. Occupancy of σ1 receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve‐injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ1 receptors attenuated the wind‐up responses in spinal cords sensitized by repetitive nociceptive stimulation. CONCLUSIONS AND IMPLICATIONS These findings contribute to evidence identifying the σ1 receptor as a modulator of activity‐induced spinal sensitization and pain hypersensitivity, and suggest σ1 receptor antagonists as potential novel treatments for neuropathic pain.