Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, and the disease is characterized by the painless nature of its lesion. Similar to leprosy, loss of pain often hinders the patients from taking proper medical care, resulting in gross deformities. A toxic lipid mycolactone produced from Mycobacterium ulcerans was thought to block the sensory system of the lesion, either by direct cellular damage (cytotoxicity) to the regional nerve tissue, or by a more sophisticated, non-toxic paralyzing mechanism. In the peripheral nerve, Schwann cells nourish axons and accelerate nerve conduction. In this study, we have compared the cytotoxic potential of mycolactone on cultured Schwann cells and that on fibroblasts, and found that mycolactone A/B induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. We demonstrated the cytotoxicity of synthetic mycolactone A/B and its remote diastereomer in mouse Schwann cells as well as fibroblasts and macrophages. A quantitative study showed that Schwann cells are relatively more sensitive than fibroblasts to mycolactone 24 and 48 hrs after exposure to mycolactone A/B diastereomer. The painless nature of Buruli ulcer may be caused by the cytotoxicity of mycolactone A/B in cultured Schwann cells.