Abstract: The aim of present study was to investigate the effect of hydroalcoholic extract of Ruta chalepensis (Rutaceae) leaves on rat ileum contractility and possible mechanism(s) involved. Ruta chalepensis extract was prepared by maceration method (ethanol 70%). Terminal portion of ileum (2 cm) was dissected out from male Wistar rats and mounted in an organ bath containing air bubbled Tyrode solution with 1 g initial tension and ileal contraction induced by KCl (60 mM) was recorded. The spasmolytic effect of the cumulative concentrations of extracts (0.01- 0.07 mg mL-1) was reduced after tissue incubation with L-NAME (100 μM, 20 min). Methylene blue (30 μM) reduced the extracts (0.01-0.07 mg mL-1) spasmolytic effect (p<0.001). Furthermore, it seems that the portion relaxatory effect of Rue extract on the rat ileum may be due to nitric oxide and the antispasmodic activity of the extract was mainly through a cGMP-dependent mechanism.