Background: 5-hydroxytryptamine 3 (5-HT3) receptors have been known to be associated with the modulation of nociceptive transmission. However, it is uncertain whether 5-HT3 plays a role in the antinociceptive or pronociceptive pathway for incisional pain. In this study, we evaluated the effects of palonosetron, a 5-HT3 receptor antagonist, on incisional pain in rats when administered intrathecally or intraplantarly. Methods: An intrathecal catheter was implanted through the cisterna magna and placed in the intrathecal space of rats. An incision in the plantaris muscle of the right hind paw was done under anesthesia with sevoflurane. Withdrawal thresholds were evaluated with the von Frey filament after 2 hours. Palonosetron (0.5 and 0.1 ${mu}g$ intrathecally; 0.5 ${mu}g$ intraplantarly) was administered and the thresholds were observed for 4 hours. Results: Mechanical hypersensitivity developed after the incision. Intrathecal palonosetron (0.5 ${mu}g$ and 0.1 ${mu}g$) did not alter the paw withdrawal threshold. Intraplantar palonosetron (0.5 ${mu}g$) also did not change the paw withdrawal threshold. Conclusions: Intrathecal and intraplantar palonosetron (0.5 ${mu}g$) had no effect on modulating the mechanical hypersensitivity in the incisional pain model of rats.