BACKGROUND: As a potential source of biomass, Jerusalem artichoke has been studied for bioethanol production; however, thus far it has not been investigated for the production of other liquid biofuels, such as biodiesel. This work aims to develop a novel approach for biodiesel production from Jerusalem artichoke tuber using heterotrophic microalgae. RESULTS: In this study, Chlorella protothecoides utilized hydrolysate of Jerusalem artichoke tuber as carbon source and accumulated lipid in vivo, with lipid content as high as 44% by dry mass, and a carbon source to lipid conversion ratio of about 25% in a 4-day scale cultivation. The lipids were extracted and then converted into biodiesel by transesterification. Cetane acid methyl ester, linoleic acid methyl ester and oleic acid methyl ester were the dominating components of the biodiesel produced. Unsaturated fatty acids methyl ester constituted over 82% of the total biodiesel content. CONCLUSION: This work suggests the feasibility of an alternative method of producing biodiesel from Jerusalem artichoke tuber using microalgae cultivation, and a cost reduction of carbon source feed in algal oil production can be expected. () 2009 Society of Chemical Industry