Destruxins (Dtxs) are insecticidal cyclic hexadepsipeptides produced by the entomopathogenic fungus Metarhizium anisopliae. Media composition for dtxs production was optimized with industrial grade media. Glycerol and casein peptone were selected as a carbon source and a nitrogen source, respectively. Dtxs production varied with C/N ratios. High yields of dtxs were observed at C/N ratios ranging 0.3 to 1.5, with concentrations mostly higher than 800 mg/L. Low yields were caused by high C/N ratio ranging from 3.0 to 8.0, resulting in less than 500 mg/L. The highest yield of Dtxs was obtained with 2% glycerol and 3% casein peptone, showing 192.2 mg/L of dtx A, 911.1 mg/L of dtx B, and 113.3 mg/L of dtx E, respectively. These results indicated that dtxs production is highly influenced by C/N ratio, especially the content of nitrogen source.