Mycolactone is a recently reported lipid toxin of Mycobacterium ulcerans that causes Buruli ulcer, a severe human skin disease. However, the mechanism of cell death by mycolactone is still unclear. In this paper, we demonstrate that mycolactone induces apoptosis in Hep 3B hepatocellular carcinoma (HCC) cells. Morphological and biochemical evidences of apoptosis, such as membrane blebbing, cell shrinkage, increase of TUNEL-positive cells and a sub-G1 cell population, were observed. To explain the mechanism of mycolactone-induced apoptosis, we examined the expression of Bcl-2 family genes. The mRNA expression of anti-apoptotic Bc1XL gene was decreased after 8 hours, while that of Mcl-1, another anti-apoptotic gene, was slowly decreased with an initial increase at second hour after treatment. Bc1-2 gene expression was extremely low both in the presence and absence of mycolactone. The expression of other Bc1-2 family genes, such as Bc1w, Bad, Bak, and Bax, was not affected. By Western blotting analysis, Mcl-1 expression (not Bc1XL) was down-regulated. Our results suggest that the down-regulation of Mcl-1 protein expression is involved in the apoptosis of Hep 3B cells by mycolactone.