Objective: The present study was undertaken to evaluate the antinociceptive effects of an ayurvedic polyherbal formulation in rats and mice employing the tail immersion test and acetic acid-induced writhing test, respectively. Materials and Methods: With the tail immersion method, rats received two different doses (270 and 405 mg/kg BW, p.o.) of a formulation, pethidine (5.4 mg/kg BW, p.o.) as a reference standard and the combination of the higher dose of the formulation with naloxone (2 mg/kg, i.p.), an opioid receptor antagonist and caffeine (16 mg/kg, i.p.), used as an adenosine receptor antagonist. In the acetic acid-induced writhing test, mice received two different doses (390 and 585 mg/kg, BW, p.o.) of formulation, diclofenac sodium (15 mg/kg, BW, p.o.) as a reference standard and the combination of the higher dose of the polyherbal formulation with ondansetron (0.5 mg/kg, i.p.), a serotonin receptor antagonist. Results: The polyherbal formulation (405 mg/kg) exhibited a significant (p < 0.01) antinociceptive effect using the tail immersion method. In the acetic acid-induced writhing test, the formulation showed significant (p < 0.01) dose-dependent activity. The antinociceptive effect of the polyherbal formulation apparently involved an opiate-like mechanism, since its antinociceptive action was attenuated by naloxone pretreatment. In addition, antinociceptive activity was attenuated by caffeine and reversed by ondansetron pretreatment. Conclusion: Our data suggest that the polyherbal formulation possessed centrally and peripherally mediated antinociceptive properties. The activity could be mediated through opioid, adenosine and serotonin receptors and via inhibition of cyclo-oxygenase- and/or lipoxygenase-dependent pathways. Copyright {\textcopyright} 2010 S. Karger AG, Basel.