The present work describes the mechanisms involved in the muscle relaxant effect of ethanol:water (40:60, 60:40 and 80:20) aerial parts extracts of Pimpinella anisum. Three hidroalcoholic extracts in which the proportion of ethanol was 40% (HA40%), 60% (HA60%) or 80% (HA80%) were tested for activity in the rat anococcygeus smooth muscle. The three extracts (50 g/mL) inhibited acetylcholine-induced contraction. The extract HA60% (5–50g/mL) concentration dependently relaxed acetylcholine-pre-contracted tissues (31.55 ± 3.56%). Conversely, HA40% and HA80% did not exert relaxant action. Pre-incubation of the preparations with NG-nitro-l-arginine methyl ester (l-NAME, 100 M), 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ, 3 M) and oxyhemoglobin (10 M) reduced the relaxation induced by HA60% (percentage of relaxation: 6.81 ± 1.86%, 13.13 ± 5.87% and 2.12 ± 1.46%, respectively). Neither indomethacin (10 M) nor tetraethylammonium (1 mM) affected the relaxation induced by HA60%. Incubation of the tissues with l-NAME significantly enhanced the maximal contraction induced by acetylcholine, indicating an inhibitory role for NO in the modulation of the contractile response of anococcygeus smooth muscle to acetylcholine. However, simultaneous addition of l-NAME and HA60% resulted in an effect similar to that observed with l-NAME alone, further confirming the observation that Pimpinella anisum acts by realizing NO. Additionally, HA60% did not alter CaCl2-induced contraction. Collectively, our results provide functional evidence that the effects elicited by the hidroalcoholic extract of Pimpinella anisum involve the participation of NO and subsequent activation of the NO-cGMP pathway. The relaxant action displayed by Pimpinella anisum justifies its use in the folk medicine as an antispasmodic agent.