High production and efficient harvesting of microalgae containing high omega-3 levels are critical concerns for industrial use. Aeration can elevate production of some microalgae by providing CO2 and O2. However, it may lower the production of others by generating shear stress, causing severe cell damage. The mixotrophic dinoflagellate Gymnodinium smaydae is a new, promising microalga for omega-3 fatty acid production owing to its high docosahexaenoic acid content, and determining optimal conditions and methods for high omega-3 fatty acid production and efficient harvest using G. smaydae is crucial for its commercial utilization. Therefore, to determine whether continuous aeration is required, we measured densities of G. smaydae and the dinoflagellate prey Heterocapsa rotundata in a 100-L semi-continuous cultivation system under no aeration and continuous aeration conditions daily for 9 days. Furthermore, to determine the optimal conditions for harvesting through centrifugation, different rotational speeds of the continuous centrifuge and different flow rates of the pump injecting G. smaydae + H. rotundata cells into the centrifuge were tested. Under continuous aeration, G. smaydaeproduction gradually decreased; however, without aeration, the production remained stable. Harvesting efficiency and the dry weights of omega-3 fatty acids of G. smaydae + H. rotundata cells at a rotational speed of 16,000 rpm were significantly higher than those at 2,000–8,000 rpm. However, these parameters did not significantly differ at injection pump flow rates of 1.0–4.0 L min-1. The results of the present study provide a basis for optimized production and harvest conditions for G. smaydae and other microalgae.