Isaria farinosa is a pathogen of alpine Thitarodes larvae that are hosts for the Chinese medicinal fungus, Ophiocordyceps sinensis. A matrix analysis indicated that the optimal culture conditions for the mycelial growth of I. farinosa are a 50-mL liquid broth in a 250-mL flask at more than 100-rpm rotation and 15-25 °C. Illumination does not affect the mycelial growth. The optimal nutrition requirements are D-(+)-galactose and D-(-)-fructose as carbon resources and D-cysteine as well as yeast powder, peptone, and beef extract as nitrogen resources at a carbon-to-nitrogen ratio of 1:1 to 1:7. The mineral component and vitamins also significantly increase the mycelial growth of I. farinosa. Based on the optimal culture conditions and nutrition requirements for the mycelial growth of I. farinosa, the effects of altitude on mycelial growth and its metabolome were evaluated using quadrupole-time-of-flight/mass spectrometry, principal component analysis and partial least squares discriminant analysis. The altitude did not affect the mycelial production but significantly regulated its metabolome. The study presents a new approach to better select a method for producing more useful metabolites and highlights the necessity of establishing standards for culturing methods related to altitude to preserve fungal quality; additionally, the results indicate that the use of a fermenter may meet the demands of large-scale mycelial production.