Abstract Objectives Bitter perception has a particularly important role in host defence. However, to date, direct effects of bitter compounds on small intestinal motility have not been shown. This study investigated the effects of bitter compounds on the spontaneous contraction of longitudinal smooth muscle strips of rat ileum. Methods Isolated longitudinal smooth muscle strips of rat ileum were used for tension recording in vitro. Immunofluorescence staining was used to identify the localization of TAS2R10 receptors. Key findings The spontaneous contraction of rat ileum was decreased after chloroquine administration. Other bitter compounds, such as quinine, denatonium and saccharin, exhibited similar effects. Chloroquine-induced relaxation was not blocked by tetrodotoxin, but was partially reversed by the nitric oxide synthase inhibitor L-NAME or the large conductance Ca2+-activated K+ (BKCa) channel antagonist iberiotoxin. By surgically removing the small intestinal mucosa or bathing in Ca2+-free Krebs solution, the chloroquine-induced relaxation was largely attenuated. The immunofluorescence staining showed that TAS2R10 receptors were expressed in rat ileum. Conclusions The results indicate that bitter receptor agonists induce relaxation of longitudinal smooth muscle strips of rat ileum, which is mediated by nitric oxide and BKCa channels.