Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.