Aims: The aim of this study is to investigate the vasorelaxant effect of 16-. O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta. Main methods: Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37°C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O2 and 5% CO2 under a resting tension of 1g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment. Key findings: ADIS (0.1μM-3mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10μM) and KCl (80mM) with intact-endothelium (Emax =79.26±3.74 and 79.88±3.79, respectively) or denuded-endothelium (Emax =88.05±3.69 and 78.22±6.86, respectively). In depolarization Ca2+-free solution, ADIS inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca2+-free medium containing EGTA (1mM) induced by PE (10μM) and caffeine (20mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5mM), 4-aminopyridine (4-AP, 1mM), glibenclamide (10μM), barium chloride (BaCl2, 1mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1μM). Significance: These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca2+ channel blocker through both intracellular and extracellular Ca2+ release.