Heliopsis longipes is used as analgesic in Mexican traditional medicine. The present study assesses the possible antinociceptive effect of Heliopsis longipes and describes the pharmacological mechanism of action of the antinociceptive effect of affinin, identified as the one active principle in Heliopsis longipes acetone extract. Intraperitoneal administration of H. longipes extract and affinin produced a dose-dependent antinociceptive effect when assessed in mice submitted to acetic acid and capsaicin tests. Affinin-induced antinociception (30 mg/kg, i. p.) was blocked by naltrexone (1 mg/kg, s. c.), p-chlorophenylalanine (80 mg/kg, i. p.) and flumazenil (5 mg/kg, s. c.) suggesting that its pharmacological effect could be due to the activation of opiodergic, serotoninergic and GABAergic systems. In addition, the antinociceptive effect of affinin was attenuated by pretreatment with 1H-[1, 2, 4] oxadiazolo [1, 2-a] quinoxalin-1-one (1 mg/kg, s. c.) and glibenclamide (10 mg/kg, s. c.) suggesting that the nitric oxide-K+ channels pathway could be involved in its mechanism of action. These results suggest that affinin itself or its derivatives may have potential antinociceptive effects.