The anti-nociceptive effect of an ethanolic leaf extract of Palisota hirsuta, a plant used locally in Ghana for various painful conditions was assessed, using various pain models. Palisota hirsuta extract (PHE) together with morphine and diclofenac (positive controls), all showed significant dose-dependent anti-nociceptive activity in all the models used, that is the tail withdrawal test, the inflammatory-induced mechanical hyperalgesia test, the acetic acid induced writhing test and the formalin test. The anti-nociceptive effect exhibited by PHE in the formalin test was reversed by the systemic administration of the non-selective opioid antagonist, naloxone, the NO synthase inhibitor, Nω-nitro-arginine methyl ester (L-NAME) and the ATP-sensitive K+ channel inhibitor, glibenclamide. However, theophyline, a non-selective adenosine receptor antagonist did not reverse this effect. PHE, unlike morphine, did not induce tolerance to its anti-nociceptive effect in the formalin test after chronic administration and also morphine tolerance did not cross-generalize to PHE. Overall, the present results demonstrate that the anti-nociceptive effects of PHE might partially or wholly be due to the stimulation of peripheral opioid receptors through the activation of the nitric oxide-cyclic GMP-ATP-sensitive K+ (NO/cGMP/K+ATP)-channel pathway without tolerance induction after chronic administration.