The transmembrane member 16A (TMEM16A)-encoded Ca(2+)-activated Cl(-) channel (CaCC) is expressed in interstitial cells of Cajal (ICCs) and involved in the generation of the slow-wave currents of gastrointestinal (GI) smooth muscles. TMEM16A modulators have been shown to positively or negatively regulate the contraction of gastrointestinal smooth muscle. Therefore, targeting the pharmacological modulation of TMEM16A may represent a novel treatment approach for gastrointestinal dysfunctions such as constipation and diarrhoea. In this study, evodiamine and rutecarpine were extracted from the traditional Chinese medicine Evodia rutaecarpa and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on intestinal peristalsis were examined. Whole-cell patch clamp results show that evodiamine and rutecarpine inhibited TMEM16A Cl(-) currents in CHO cells. The half-maximal inhibition values (IC(50)) of evodiamine and rutecarpine on TMEM16A Cl(-) currents were 11.8 +/- 1.3 muMu and 9.2 +/- 0.4 muM, and the maximal effect values (E(max)) were 95.8 +/- 5.1% and 99.1 +/- 1.6%, respectively. The Lys(384), Thr(385), and Met(524) in TMEM16A are critical for evodiamine and rutecarpine's inhibitory effects. Further functional studies show that both evodiamine and rutecarpine can significantly suppress the peristalsis in isolated guinea-pig ileum. These findings demonstrate that evodiamine and rutecarpine are new TMEM16A inhibitors and support the regulation effect of TMEM16A modulators on gastrointestinal motility. CI - Copyright (c) 2021. Published by Elsevier B.V.