Abstract Salvia divinorum is a widespread hallucinogenic herb traditionally employed for divination, as well as a medicament for several disorders including disturbances of gastrointestinal motility. In the present study we evaluated the effect of a standardized extract from the leaves of S. divinorum (SDE) on enteric cholinergic transmission in the guinea-pig ileum. SDE reduced electrically evoked contractions without modifying the contractions elicited by exogenous acetylcholine, thus suggesting a prejunctional site of action. The inhibitory effect of SDE on twitch response was abolished by the opioid receptor antagonist naloxone and by the κ-opioid antagonist nor-binaltorphimine, but not by naltrindole (a δ-opioid receptor antagonist), CTOP (a μ-opioid receptor antagonist), thioperamide (a H3 receptor antagonist), yohimbine (an α2-receptor antagonist), methysergide (a 5-hydroxytryptamine receptor antagonist), NG-nitro-l-arginine methyl ester (an inhibitor of NO synthase) or apamin (a blocker of Ca2+-activated K+ channels). Salvinorin A, the main active ingredient of S. divinorum, inhibited in a nor-binaltorphimine- and naloxone-sensitive manner electrically induced contractions. It is concluded that SDE depressed enteric cholinergic transmission likely through activation of κ-opioid receptors and this may provide the pharmacological basis underlying its traditional antidiarrhoeal use. Salvinorin A might be the chemical ingredient responsible for this activity.