Shatavari (Asparagus racemosus) is a traditional Ayurvedic herb and is often used for infertility and for women’s health. Animal studies have been conducted in vivo and demonstrated that Shatavari may have tocolytic effects. However, the direct effects of Shatavari on the myometrium and its mechanisms of action have never been clarified. As relaxants of the myometrium are needed to help reduce dysmenorrhea, miscarriage and preterm labour, the aims of the present study were therefore, to determine the effects on the myometrium of Shatavari root extract and to investigate its mechanisms of action. Shatavari roots were ethanolic extracted. Non-pregnant and pregnant rats were humanely killed by asphyxiation with CO2 and longitudinal uterine smooth muscles 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 Shatavari root extract studied. The effects of Shatavari root extract on spontaneous, high-K-depolarisation-induced and oxytocin-induced contraction in both non-pregnant and pregnant rats were particularly examined. The results show that Shatavari root extract significantly decreased force produced by all three mechanisms, in a dose dependent manner. However, the effects of the extract in pregnant rats were more potent compared to those of non-pregnant rats. The extract also significantly decreased force when oxytocin was added under depolarised conditions and when force was produced only by sarcoplasmic reticulum Ca release. Thus these data show that Shatavari is a potent uterine relaxant, decreasing force produced by both Ca-dependent and -independent pathways. The inhibition of uterine activity of Shatavari may be a useful source of uterine relaxant and its active ingredient on the uterus should be identified, as it may be helpful for dysmenorrhea, miscarriage and preterm labour.