Cordyceps unilateralis, a fastidious insect pathogenic fungus, was found to produce red naphthoqinones, which possess anti-malarial activity. A method for the isolation and in vitro cultivation of this fungus was, therefore, developed in this study. Ascospores from ant cadavers were used as starting materials, and their secondary spores were allowed to germinate first on PDA for 2 d. Secondary spores were then transferred to Grace's Insect Cell Medium+2 mM glutamine and 10% fetal bovine serum, incubated for 7 d, after which the formation of mycelia was stimulated by transfer to potato dextrose broth. As a result, the growth of C. unilateralis from the steps of secondary spore germination, blastospore formation and mycelial growth was successfully completed for the first time.