γ-Aminobutyric acid (GABA) receptor/channel ρ1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel ρ1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel ρ1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel ρ1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel ρ1 subunit mutant mice (rho1-/-). GABA receptor/channel ρ1 subunit expression in the rho1 -/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho1-/- mice. A decreased threshold for mechanical pain in the rho1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel ρ1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.