Fluoxetine (FLX), a well-known antidepressant drug under the class of selective serotonin reuptake inhibitor, exerts its action by inhibiting the reuptake of serotonin selectively. In some studies, it has been demonstrated that FLX relaxes the intestinal smooth muscle. In this study, we aimed at studying the signal transduction pathway underlying the muscle relaxation effect of FLX on electrically stimulated rat ileal muscle contraction. To investigate the possible mechanism involved, various antagonists were used. It was found that inhibition with L-N-G-nitroarginine methyl ester, ondansetron, GR113808 and bicuculline enhanced the relaxation effect of FLX. However, the effect of FLX was nullified under the presence of atropine, calcium channel modulator (calcium ionophore A23187), and potassium channel blockers (tetraethylammonium chloride, 4-aminopyridine and glybenclamide). Specific pathway-inhibiting antagonists, Y27632 (Rho-kinase inhibitor) and U73122 (phospholipase-C inhibitor) reversed the antagonistic effect of FLX, while ML-9 (myosin light chain kinase inhibitor) and chelerythrine (protein kinase C inhibitor) augmented the FLX-induced inhibition effect. Taken together, we concluded that FLX exerts the inhibitory effect on electric field stimulation response in rat ileal smooth muscle by the inhibition of muscarinic receptors, decrease of intracellular calcium level by inhibiting phospholipase C and opens the potassium channels. (C) 2017 S. Karger AG, Basel