We have previously verified that niga-ichigoside F1 (NI), a triterpene isolated from Rubus imperialis, exhibits significant and potent antinociceptive action when evaluated in some pharmacological models of pain in mice. This effect was confirmed in other experimental models and also the mechanism of action has been evaluated. The antinociception caused by NI (60 mg kg−1) in both phases of the formalin test was significantly attenuated by intraperitoneal injection of mice with haloperidol (a dopaminergic antagonist, 0.20 mg kg−1) and L-arginine (precursor of nitric oxide, 600 mg kg−1). Regarding the cholinergic system, atropine (a cholinergic antagonist 60 mg kg−1) reverted only the second phase. The effect of NI was not affected by treatment of mice with yohimbine (an alpha2-adrenoceptor antagonist, 0.15 mg kg−1). The same pharmacological profile was observed for the administration of naloxone (an opioid receptor antagonist, 1 mg kg−1). On the other hand, intraperitoneal injection caused dose-related and significant effects against glutamate- and capsaicin-induced pain, respectively. In conclusion, the marked antinociception of NI appears to be related to the dopaminergic, cholinergic, glutamatergic, tachykininergic and oxinitrergic systems, supporting the ethnomedical use of Rubus imperialis (Rosaceae).