Aim of the study: The effects of pomegranate juice and its components on uterine smooth muscle are unknown. Hence, this study aimed to determine the effects of pomegranate juice on the myometrium and investigate their mechanisms of action.Methods Rat uterine smooth muscle horn strips were suspended in organ baths containing Krebs solution. Contractions were measured isometrically using a transducer (AD instrument Australia). The effects of pomegranate juice were evaluated on contractile activity elicited by potassium chloride (KCl 60Mm) depolarization. Inhibitors of nitric oxide (L-NAME 3X10− 4), guanylate cyclase (methylene blue 1*10− 5) and Prostaglandin I2 (indomethacin 3X10− 5); in addition, Potassium Channels blockers, as well as calcium signaling pathways, were determined.Results The juice at concentrations from 1.5-5 mg/ml significantly decreased the rat uterine horn contraction induced by KCl. The inhibitors of NO, cGMP, and PGI2 produced no blocking effect on the relaxation response. Furthermore, the PGI2 inhibitor significantly enhanced the relaxation effects; K + channel blockers did not have any inhibitory effects on the relaxation responses. Contrarily, GLIB significantly improved relaxation. The L-type Ca2 + channel blocker prevented spontaneous contraction of the uterine.Conclusion These findings imply that the active ingredient in pomegranate juice can be used to reduce uterine contractions. Pomegranate juice's action demonstrated that it could aid in treating disorders with uterotonic effects. It may be useful in preventing preterm birth and menstrual cramps, but more research is needed to determine the mechanism of action.Trial registration Code: AEC-013. Registered 23 October 2015.