Lysteriolysin O (LLO) induces a microtubule‐dependent activation of mucin exocytosis in the human mucin‐secreting HT29‐MTX. Cholesterol inhibits the LLO‐induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO‐induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29‐MTX cells results in a decrease in the LLO‐induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO‐induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane‐associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO‐induced mucin exocytosis develops independently of the pore‐forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent‐insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.