Background: Vitamin D receptors (VDR) are expressed in many reproductive tissues indicating a potential role of vitamin D3-VDR in the regulation of reproductive functions. Nevertheless, the data about the effect of vitamin D3 in the uterus are scarce. Aim of the Study: This study was designed to evaluate the potential effects of vitamin D3 on spontaneous, KCl- induced and oxytocin-induced contractions in the rat uterus in vitro. Material and Methods: The study was conducted on 10 healthy adult female albino non-pregnant rats. Full-thickness longitudinal muscle strips were dissected from each non- pregnant rat and then myometrial tension was recorded. The strips were mounted vertically in organ baths and exposed to vitamin D3 and different uterotonic agents to delineate the Potential action of vitamin D3 on the rat myometrial contractility. Spontaneous contractions were recorded using mechanical activity recording system. We evaluated the effects of 3 different doses of vitamin D3 on spontaneous uterine contrac- tions; then on concentrated KCl-induced uterine contractions and oxytocin (OT)-induced uterine contractions. Furthermore, the effects of vitamin D3 on spontaneous uterine contractions pretreated with nifedipine; a voltage-gated L-type Ca2+ channel antagonist were investigated. Results: Vitamin D3 significantly inhibited the spontaneous uterine contractions in a dose dependent manner. Moreover, vitamin D3 inhibited the uterine contractions whether induced by oxytocin or concentrated KCl. Administration of nifedipine resulted in a significant decline in the force amplitude of spontaneous uterine contractions. In the presence of vitamin D3, the uterine relaxant effect of nifedipine was significantly augmented. Conclusions: The inhibitory effects of the in vitro administration of vitamin D3 on rat uterus may yield novel insights into its therapeutic use. Further, it may be recommended to be used as a novel tocolytic agent for preventing unwanted uterine contractions in early pregnancy and relieving pain related to dysmenorrhea