The noradrenalin and serotonin re-uptake inhibitor venlafaxine has an analgesic effect that is independent of its antidepressant activity; however, the mechanism of this effect remains to be elucidated. This study was performed to investigate the possible roles of the opioidergic system and nitric oxide (NO) pathway in the analgesic effect of venlafaxine. Eighty Wistar rats of both sexes were allocated to 10 groups. The hot plate test was used to assess the antinociceptive/analgesic effect. The temperature of the hot plate was adjusted to 52.5±1°C, the cut-off period was set to be 50 sec; licking of the hind paw was used as a sign of pain perception. Venlafaxine alone (25 mg/kg) showed marked analgesic activity (p<0.05). N-ω-nitro-L-arginine (L-NOARG) alone (20 mg/kg) and naloxone alone (2 mg/kg and 4 mg/kg) showed no analgesic activity (p>0.05). Coadministration of low-dose naloxone (2 mg/kg) and both doses of L-NOARG (20 and 40 mg/kg) with venlafaxine (25 mg/kg) did not modify the analgesic effect but high-dose naloxone (4 mg/kg) decreased it significantly (p<0.05). In conclusion, these results suggest that the opioidergic system but not the NO pathway has a role in the analgesic effect of venlafaxine.