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Abstract: Background: The extraneuronal cholinergic system has been implicated in numerous functions in the skin, such as terminal differentiation, barrier formation, sweat secretion and the microcirculation. However, the evidence for cholinergic signalling in sebaceous glands is lacking, and its role needs to be clarified. Objective: We investigated the role of acetylcholine signalling in sebaceous glands using human sebocytes and a clinical study using botulinum toxin. Methods: Immunohistochemistry and immunocytofluorescence were performed to evaluate cholinergic receptor levels in sebaceous glands. Lipid levels were assessed by Oil Red O staining and signalling pathways by Western blotting. To evaluate the clinical relevance, we also assessed the effect of botulinum toxin on sebum production in healthy volunteers. Results: We demonstrated that human skin sebaceous glands in vivo and sebocytes in vitro express nicotinic acetylcholine receptor α7 (nAchRα7), and that acetylcholine increased lipid synthesis in a dose-dependent manner. When sebocytes were incubated with α-bungarotoxin, a competitive nAchR antagonist, acetylcholine failed to up-regulate lipid synthesis. Twenty healthy volunteers were enrolled in a double-blind, placebo-controlled, split-face study. A marked decrease in sebum production on the botulinum-treated side was found in volunteers with oily skin. Conclusion: These results provide evidence that acetylcholine signalling plays a significant role in human sebaceous gland biology and identify acetylcholine signalling as a promising target in the clinical management of disorders in which sebum production is increased, such as acne vulgaris. [Copyright &y& Elsevier]