Annona leptopetala R. E. Fries (basynonymy Rollinia leptopetala R. E. Fries) is an endemic tree or shrub from Brazil. Some Annona species such as Annona muricata and Annona squamosa showed uterus activities. Thus, in the search for new agents to combat the uterine disorders, it was aimed to investigate a possible AL-EO tocolytic effect on isolated rat uterus and the underlying mechanisms. Uterine horns were removed, cleaned of adhering fat and connective tissue, and suspended by in organ baths containing Locke-Ringer solution, bubbled with carbogen mixture and the contractions were registered using a force transducer. All experimental procedures were previously approved by Ethic Comission on Animal Use of UFPB (protocol n° 0104/2014). AL-EO was more potent to relax pre-contracted uterus with oxytocin than with KCl. Moreover, AL-EO shifted to the right, in a non-parallel manner, oxytocin‑induced cumulative contraction curves, with E_max reduction, indicating a pseudo-irreversible noncompetitive antagonism of oxytocin receptors. In the presence of propranolol, β-adrenergic antagonist, an AL-EO action increase was observed. Nitric oxide and cyclooxygenase pathways participation were assessed and discarded. The tocolytic potency of AL-EO was attenuated by CsCl, a non-selective K⁺ channel blocker, suggesting a positive modulation of these channels. Additionally, in the presence of 4-aminopyridine (4-AP) but not in apamin, glibenclamide or tetraethylammonium (1 mM), the relaxant potency of AL-EO was reduced, indicating the voltage-dependent potassium channels participation, but not small- or big- conductance Ca²⁺-activated and ATP‑sensitive potassium channels. Tocolytic mechanism of AL-EO on rat uterus involves pseudo-irreversible noncompetitive antagonism of oxytocin receptor and β‑adrenergic receptors and positive modulation of voltage-dependent potassium channels. Thus, AL-EO is presented as a promising drug with activity on uterine conditions, as dysmenorrhea, and after further evaluation in clinical studies, it would be used as an alternative drug on current pharmacotherapy.