Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in Guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10-7 M) than by KCl (EC50=2.6±0.5×10-6 M) or carbachol (EC50=1.8±0.4×10-6 M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca2+-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10-6 M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10-6 M), but apamin or tetraethylammonium (1 mM) had no effect (EC50=2.6±0.2×10-7 and 1.6±0.3×10-7 M, respectively), ruling out the involvement of small and big conductance Ca2+-activated K+ channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10-6 and 1.5±0.5×10-6 M, respectively), indicating the involvement of voltage- and ATP-sensitive K+ channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca2+ concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca2+ levels.