Effect of oxygen supply on cordycepin production was investigated in submerged cultivation of Cordyceps militaris, a famous traditional Chinese medicinal mushroom, in a 5‐L turbine‐agitated bioreactor (TAB). Initial volumetric oxygen transfer coefficient ( kLa) within the range of 11.5–113.8 h−1 had significant influence on cordycepin production. The highest cordycepin concentration of 167.5 mg/L was obtained at an initial kLa value of 54.5 h−1, where a moderate dissolved oxygen (DO) pattern was observed throughout cultivation. The possible correlation between cordycepin production and DO level was explored by DO control experiments, and the results showed that DO within the range of 10–80% of air saturation greatly affected the cultivation process. To obtain a high specific cordycepin formation rate (ρ) throughout cultivation, a two‐stage DO control strategy was developed based on the analysis of the relationship of ρ and DO. That is, DO was controlled at 60% from the beginning of cultivation and then shifted to a lower control level of 30% when ρ started to decrease. As a result, a high cordycepin production of 201.1 mg/L and a high productivity of 15.5 mg/(L·d) were achieved, which was enhanced by about 15% and 30% compared to the highest titers obtained in conventional DO control experiments, respectively. The proposed DO control strategy was also applied to a recently developed 5‐L centrifugal impeller bioreactor (CIB) with cordycepin production and productivity titers of 188.3 mg/L and 14.5 mg/(L·d). Furthermore, the scale‐up of the two‐stage DO control process from 5‐L CIB to 30‐L CIB was successfully demonstrated. The work is useful for the efficient large‐scale production of bioactive metabolites by mushroom cultures.