A method of culturing a microorganism capable of producing at least any one of a highly unsaturated fatty acid or a compound containing a highly unsaturated fatty acid as a constituent fatty acid in a culture medium containing at least a carbon source and a nitrogen source, using an aeration-agitation culture device capable of adjusting and controlling an agitation power and an aeration amount. The method comprises the steps of performing mechanical agitation for a predetermined time after start of culture, where the agitation power per unit liquid amount is 269 (W/m3) or less, and after the predetermined time has passed, adjusting and controlling at least any one of the maximum aeration amount and a maximum power required for agitation to a range which satisfies that KLA (=(P/V)0.95Vs0.67) is 59 or more, an air flow rate parameter Vs0.67 is 0.075 or more, and a required agitation power parameter (P/V)0.95 is 203 or more, where P represents power required for agitation (W), V represents a liquid amount (m3), and Vs represents an air flow rate (m/sec).