Alpha-2A adrenergic receptor (AR) subtype plays an important role in the analgesic effect of α2-AR agonists. Here, we examined the effects of α2-AR agonists, dexmedetomidine and xylazine, on spinal synaptic transmission in newborn C57BL/6J and α2A-AR mutant mice. Spinal reflex potentials, the monosynaptic reflex potential (MSR) and the slow ventral root potential (sVRP), were measured in isolated spinal cords. The compound action potential was measured in isolated lumbar nerve. Dexmedetomidine and xylazine suppressed both the MSR and sVRP in a concentration-dependent manner. In α2A-AR mutant mice, sVRP suppression by dexmedetomidine was greatly weakened, while that by xylazine (30-100μM) showed only slight attenuation. A high concentration (300μM) of xylazine completely suppressed the sVRP, even in α2A-AR mutant mice spinal cords, and also suppressed the compound action potential. MSR suppression by these α2-AR agonists had no difference between wild-type and α2A-AR mutant mice. These results suggest that sVRP suppression by dexmedetomidine and xylazine is mainly mediated by α2A-AR. In addition, a high concentration of xylazine inhibits conduction of the action potential, which is not mediated by α2A-AR. α2-AR is not responsible for the dexmedetomidine- and xylazine-mediated inhibition of the MSR.