The l-arginine/nitric oxide/cyclic GMP pathway has been proposed as the mechanism of action for peripheral antinociception concerning several groups of drugs, including opioids and nonsteroidal analgesics. The aim of the present study was to investigate the involvement of the l-arginine/NO/cGMP pathway on antinociception induced by xylazine, an α2-adrenoceptor agonist extensively used in veterinary medicine and animal experimentation. The rat paw pressure test was used by inducing hyperalgesia via intraplantar injection of prostaglandin E2 (2 μg). Xylazine was administered locally into the right hind paw (25, 50 and 100 μg) and either NO synthase inhibitor L-NOarg (12, 18 and 24 μg/paw), soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100 μg/paw) or cGMP-phosphodiesterase inhibitor zaprinast (50 μg/paw) were previously administered to the right hind paw of Wistar rats. Xylazine administration elicited a local antinociceptive effect, since only much higher doses produce a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine (100 μg/paw) was antagonized by L-NOarg and by ODQ; however, zaprinast potentiated the antinociceptive effect of xylazine at 25 μg/paw. The results provide evidence that xylazine probably induces peripheral antinociceptive effect by l-arginine/NO/cGMP pathway activation.