Watermelon (Citrullus lanatus) is a natural and rich source of the amino acid citrulline; a precursor of L-arginine, the substrate for nitric oxide synthase in the production of NO. Recent work has shown that watermelon consumption increases plasma arginine in adults and that increased watermelon ingestion improved aortic haemodynamics. While several studies have also examined the in vitro effects of citrulline on vascular smooth muscle, there appears to be no studies of its effect on the myometrium. As relaxants of the myometrium are needed to help reduce preterm deliveries, the aims of the present study were therefore, to determine the effects on the myometrium of watermelon extracts and to investigate their mechanisms of action. Watermelon flesh and rind were ethanolic extracted. Rats were humanely killed with CO2, and longitudinal uterine smooth muscle was dissected. The animal procedures were conducted in accordance with the Institutional Animal Care and Use Committee, Suranaree University of Technology, Thailand. Isometric force was measured and the effects of extracts of watermelon flesh and rind were evaluated on three types of contractile activity; spontaneous, those elicited by KCl depolarization (40 mM) or oxytocin (10 nM) application. Inhibitors of NO and their mechanisms of action, L-NAME (100 μM), LY83583 (1 μM), and tetraethylamonium chloride (5 mM), as well as Ca signaling pathways, were determined. The results show that both flesh and rind extracts significantly decreased force produced by all three mechanisms, in a dose dependent manner. The extracts could also significantly decrease force under conditions of sustained, high Ca levels (depolarization and agonist) and when force was produced only by sarcoplasmic reticulum (SR) Ca release. Investigation of L-citrulline (64 μM) demonstrated that it produced the same effects on force as watermelon extracts. Combination of L-citrulline and watermelon extracts, at their EC50 doses produced additive effects. The inhibitory effects of extracts and L-citrulline were reversed upon addition of the inhibitors of NO, and pretreatment of tissues with these inhibitors prevented the actions of both extracts and L-citrulline. Thus these data show that watermelon is a potent tocolytic, decreasing force produced by calcium entry and SR release and arising by different pathways, including oxytocin stimulation. Its major mechanism appears to be to stimulate the NO-cGMP relaxant pathway, which can be accounted for by its constituent, citrulline.