As a naturally occurring flavone, luteolin has received much attention due to its antioxidant, anti-inflammatory and anticancer functions. In the present study, we investigated the effect of luteolin on colonic motility and its mechanism using isometric muscle recording and the whole-cell patch-clamp technique in mice. Luteolin dose-dependently inhibited colonic smooth muscles motility and CMMC significantly. BayK8644, an L-type Ca2+ channel agonist, significantly attenuated the luteolin-induced inhibition. Moreover, the calcium currents recorded in colonic smooth muscle cells were dramatically inhibited by luteolin. However, no significant changes were found in the luteolin-induced inhibitory effect in the presence of TEA, a nonselective K+ channel blocker, glibenclamide, an ATP-dependent K+ channel blocker, and apamin, a small-conductance Ca2+-activated K+ channel blocker. Additionally, luteolin did not affect potassium currents. Furthermore, TTX, a Na+ channel blocker, L-NAME, an inhibitor of nitric oxide (NO) synthase, ODQ, an inhibitor of NO-sensitive guanylyl cyclase, and Ani9, a specific ANO1 channels blocker, had no effect on the luteolin-induced suppression. These results suggest that luteolin inhibited colonic smooth muscle motility by inhibiting L-type calcium channels in mice but not through potassium channels, the enteric nervous system (ENS), NO signaling pathways or ANO1 channels of interstitial cells of Cajal (ICCs).