In the present study, the antinociceptive profile of oligopeptidases B from Trypanosoma cruzi (OPTc) and Trypanosoma brucei (OPTb) were examined in mice evaluated by the acetic acid-induced writhing test. Both OPTc and OPTb injected intraperitoneally attenuated the writhing numbers in the acetic acid-induced writhing test. This effect was not dependent on the enzymatic activity, but the enzyme structure was important for this purpose. Intraperitoneal pretreatment with methysergide (5-HT serotonergic receptor antagonist) attenuated antinociceptive effect induced by both OPTc and OPTb in the writhing test. However, naloxone (opioid receptor antagonist) or yohimbine (α2-adrenergic receptor antagonist) did not affect antinociception induced by both oligopeptidases. Our results suggest that OPTc and OPTb show antinociceptive property in the writhing test. Furthermore, this antinociceptive effect may be mediated by serotonergic receptor but not opioidergic or α2-adrenergic receptors.