Pereskia aculeata Miller (Cactaceae) is a cactus distributed from south to northeast of Brazil, where its leaves are commonly used as a vegetable, in skin wound healing, and to treat inflammation. The objective of this study was to perform the chemical characterization and to evaluate the antinociceptive activity of the hydromethanolic fraction obtained from the methanol extract of P. aculeata leaves. Chemical characterization was performed by UPLC-MS analysis. The antinociceptive activity was evaluated by the acetic acid-induced writhing, formalin, and tail-flick tests in mice, administering the single oral doses of 100, 200, and 300 mg/kg 1 h before each test. Tryptamine, abrine, mescaline, hordenine, petunidin, di-tert-butylphenol isomers, and quercetin were identified. The antinociceptive activity was inversely proportional to the administered doses in the acetic acid test, as the dose of 100 mg/kg reduced by 78% the number of writhings, while the doses of 200 and 300 mg/kg reduced by 64% and 41%, respectively. In the formalin test, the dose of 300 mg/kg inhibited by 50% and 86% the licking paw time in the first and second phases, respectively, while the doses of 200 mg/kg (45% and 62%, respectively) and 100 mg/kg (15% and 48%, respectively) were less effective. The sample did not respond to the tail-flick test. Those results suggested a peripheral and central antinociception devoid of an opioid effect. Pereskia aculeata not only is a plant food with high nutritional value but also presents analgesic potential. It is the first time that this bioactivity is reported for this species.