Schizochytrium limacinum SR21 is an important strain for industrial production of docosahexaenoic acid (DHA), which is an important omega-3 fatty acid used in the nutraceutical and food industry. However, the high cost of carbon sources has limited its further application in the market with much larger volume, such as animal feed for aquaculture, poultry, and livestock. To seek low-cost carbon source, acetic acid is tested in the present study. The effect of different factors, including initial carbon source concentration, pH, aeration rate, and nitrogen sources, on biomass, lipid, and DHA production were tested. With optimized culture conditions, the biomass concentration of 146 g/L, total fatty acids (TFAs) of 82.3 g/L, and DHA content of 23.0 g/L were achieved with a pH-auxostat fed-batch cultivation. These results suggested that acetic acid is a promising feedstock for the low-cost production of DHA.