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Mushroom is an abundant source of a wide range of useful natural products with biological activities. Since the field cultivation of mushroom takes several months to yield the fruiting body with a low productivity of bioactive compounds, submerged cultivation of mushrooms is viewed as a promising alterative for producing valuable substances. Cordyceps militaris, a caterpillar-shaped Chinese traditional medicinal mushroom, is an entomopathogenic fungus, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs. Besides its usage as a crude drug, it has been extensively used as folk tonic food or invigorant since ancient times. This mushroom produces an important bioactive compound, cordycepin (3′-deoxyadenosine), which is a nucleoside analogue. Cordycepin is reported to possess many interesting biological and pharmacological activities, including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. Previous work reported the isolation of cordycepin from liquid culture medium of C. militaris and its pharmacological functions. Trace levels of cordycepin were mentioned in mycelium and culture broth during submerged cultivation of Cordyceps sp. in potato-dextrose medium. Jia et al. reported that 7.1 mg cordycepin/l was acquired in an airlift bioreactor cultivation of C. militaris. However, there have been no reports on dynamic profiles of cordycepin production by submerged cultivation of C. militaris, and the effect of medium nutrients has not been revealed as yet. Usually, culture medium is important to the yield of mycelia, and medium composition generally play a significant role because these nutrients are directly linked with cell proliferation and metabolite biosynthesis. Various statistical experimental design strategies were applied to the optimization of submerged medium such as for lovastatin production by Monascus rubber. But, as far as we know, there is limited knowledge about the nutritional requirement for cordycepin production by C. militaris, and there have been no reports on medium optimization to improve cordycepin production. In this work, the effects of medium composition were focused in order to improve the cordycepin production by submerged cultivation of C. militaris. The information obtained is considered fundamental and useful to the development of C. militaris cultivation process for efficient production of cordycepin on a large scale.