Cordycepin, a beneficial bioactive product specifically found in Cordyceps, has received attention in various bioindustrial applications such as in pharmaceuticals, functional foods, and cosmetics, due to its significant functions. However, low productivity of cordycepin is a barrier to commercialization. In this study, Cordyceps militaris was mutated by UV irradiation to improve the cordycepin production. The highest producer KYL05 strain was finally selected and its cordycepin production was increased about 1.5-fold compared to wild type. In addition, the effects of culture conditions were fundamentally investigated. Optimal conditions were as follows: pH 6, temperature of 25 °C, shaking speed of 150 rpm, and culture time of 6 days. Effects of medium component on cordycepin production were also investigated by using various carbon and nitrogen sources. It was found that glucose and casein hydrolysate (CH) were most effective as carbon and nitrogen sources in cordycepin production (2.3-fold improvement) with maximum cordycepin production of about 445 mg/L. In particular, production was significantly affected by CH. These results should be of value in improving the efficiency of mass production of cordycepin.