Environmental and medium factors were investigated as basic data for optimizing DHA production when using Thraustochytrium aureum. To study the effect of environmental conditions, the rotation speed and culture temperature were changed. Plus the trend of the growth characteristics, lipid content in the biomass, and DHA content in lipids were evaluated according to various initial glucose concentrations. The biomass, lipid, and DHA analyses showed that the physiological characteristics of T. aureum were closely related with the environmental and medium conditions, as in the case of other marine microorganisms. For example, a low rotation speed of 50 rpm lowered the cell growth rate as well as the DHA content in the lipids. A low temperature had a negative effect on the cell growth, yet a positive effect on the lipid content in the biomass. Different initial glucose concentrations had no effect on the lipid content in the biomass or DHA content in the lipids, yet did affect the cell growth. Accordingly, these results show that environmental and medium factors must be synthetically considered in order to optimize DHA production when using T. aureum.