The ethanolic extract from Croton blanchetianus leaves has been shown to have antinociceptive activity in mice. Here, we investigated the antinociceptive activity of an ethyl acetate fraction (EAF) from this extract in mice and the possible pathways involved in the analgesic effect. Adverse effects on behavior and motor coordination were also evaluated. The EAF was characterized by liquid chromatography coupled with mass spectrometry and evaluated (12.5, 25, and 50 mg/kg per os) in the acetic acid-induced abdominal writhing, formalin, hot plate, and tail immersion assays. Naloxone, atropine, glibenclamide, prazosin, or yohimbine was pre-administered to mice to investigate the involved pathways in the formalin test. The open-field, rotarod, and elevated plus-maze tests were used to assess behavior and locomotion. The main components of the EAF were quercetin-3-O-(2-rhamnosyl) rutinoside, hyperoside, quercetin rutinoside pentoside, and quercetin hexoside deoxyhexoside. EAF showed antinociceptive effects in all models and was effective against both neurogenic and inflammatory pain. The reversion of the effects in the formalin test by naloxone and atropine revealed that the EAF acted via the opioid and cholinergic systems. In the open-field test, the behavior of the animals treated with the EAF was like that of control, except at the highest dose, when hypnosis, eyelid ptosis, decreased walking, hygiene, and rearing behaviors were observed. No muscle relaxant effect was observed, but an anxiogenic effect was observed at all doses. This study provides new scientific evidence on the pharmacological properties of C. blanchetianus leaves and their potential for the development of phytomedicines with analgesic properties.