In the present study, spent yeast from a brewery was used as the growth substrate for the docosahexaenoic acid (DHA)-rich microalga, Aurantiochytrium sp. KRS101. A significant biomass yield (6.69 g/l/d) was obtained using only spent yeast as the growth substrate, with simple stirring as pretreatment. Maximization of nutrient utilization through the use of stepwise cultivation increased the yield to 31.8 g/l of biomass. DHA constituted 38.2% (w/w) of the total fatty acids, and the highest DHA productivity was observed when the C/N ratio was 20:1 (w/w). Spent yeast thus served as a good growth substrate for the production of DHA. Economic assessment revealed that stepwise cultivation using spent yeast as either the sole growth substrate or as a nutrient source could substantially reduce the production cost of microalgal DHA. (C) 2012 Elsevier Ltd. All rights reserved.