The anticonvulsant effects of agmatine, an endogenous polyamine and a metabolite of l-arginine, have been shown in various experimental seizure models. Agmatine also potentiates the anti-seizure activity of morphine. The present study aimed to investigate a possible involvement of nitric oxide (NO) pathway in the protection by agmatine and morphine co-administration against pentylenetetrazole (PTZ) –induced seizure in male mice. To this end, the thresholds for the clonic seizures induced by the intravenous administration of PTZ, a GABA antagonist, were assessed. Intraperitoneal administration of morphine at lower dose (1 mg/kg) increased the seizure threshold. Also intraperitoneal administration of agmatine (5 and 10 mg/kg) increased the seizure threshold significantly. Combination of subeffective doses of morphine and agmatine led to potent anticonvulsant effects. Non-effective doses of morphine (0.1 and 0.5 mg/kg) were able to induce anticonvulsant effects in mice pretreated with agmatine (3 mg/kg). Concomitant administration of either the non-selective nitric oxide synthase (NOS) inhibitor L-NAME (1, 5 mg/kg, i.p.) or the selective NOS inhibitor 7-NI (15, 30 mg/kg, i.p.), with an ineffective combination of morphine (0.1 mg/kg) plus agmatine (1 mg/kg) produced significant anticonvulsant impacts. Moreover, the NO precursor, l-arginine (30, 60 mg/kg, i.p.), inhibited the anticonvulsant action of agmatine (3 mg/kg) plus morphine (0.5 mg/kg) co-administration. Our results indicate that pretreatment of animals with agmatine enhances the anticonvulsant effects of morphine via a mechanism which may involve the NO pathway.