In this study, acid-catalyzed hot-water extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. was performed, and its yield-enhancing effects were investigated. The total fatty acid content of the Aurantiochytrium sp. was 482.5 mg/g cell, of which 141.7 mg/g cell (29.4% of total fatty acids) was DHA. The lipid-extraction yield by acid-catalyzed hot-water treatment was compared with those by organic solvents. Among the various acid-catalyzed hot-water treatment conditions, the most optimal were 1.00% H2SO4 concentration, 100 degrees C, 30 min, under which the lipid-extraction yield was 472.4 mg/g cell, and most of the DHA was extracted (29.2% of total fatty acids). Acid-catalyzed hot-water extraction treatment markedly improved the lipid-extraction yield of Aurantiochytrium sp.