Aim: To explore the emodin’s toxicity and action mechanism on the function of interstitial cells of Cajal (ICC) cultured in vitro. Methods: ICC of KM mouse was cultured in vitro. The minimum toxicity concentration and critical time points of emodin were investigated with Uniform Design methodology and MTT assay. The cell enzymology assay and enzyme immunoassay (EIA) were applied to observe the effect of emodin on membrane stability, cellular internal environment, energy metabolism and second messenger of ICC. Results: The minimum toxicity concentration was 0.001%, and the critical time points were 30 s, 1 min, 30 min, and 60 min. After administration of emodin, the damage on cells aggravated with time prolonging. The activity of malonaldehyde (MDA), lactate dehydrogenase (LDH), and phosphatase in the cell was raised significantly (P < 0.01). The concentrations of Na⁺ and Ca²⁺ were increased but K⁺ concentration was decreased. The Na⁺-K⁺-ATPase activity was promoted but Ca²⁺-ATPase descended. Second messenger as IP3 and cAMP also became more active. All these changes had statistical significance (P < 0.01). Conclusion: Emodin had toxicity function on ICC which can lead to membrane damage, energy metabolism disorder. This mechanism could be related to electrolytes concentration disorder, inhibited activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase, and raised activity of IP3 and cAMP.