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Aims:  To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called ‘ion beam’. Methods and Results:  Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8‐azaadenine‐ and 28 classes of 8‐azaaguanine‐resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8‐azaadenine‐resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8‐azaaguanine‐resistant mutants, it was shown that mutant no. G81‐3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3·1 and 1·8 g l−1 on 21‐day culture for mutant no. G81‐3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. Conclusions:  The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. Significance and Impact of the Study:  The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large‐scale production of cordycepin.

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