Background : The purpose of the present investigation is to enhance extracellular acidic protease production by subjecting a protease producing strain Cordyceps pruinosa DK-01 to random mutagenesis by UV irradiation after ethidium bromide treatment. Methods and Results : Mutants were screened as protease producers on the basis of zone of clearance and relative proteolytic activity (RPA) on skimmed milk agar plates. In addition, mutants showed strong pink-red color intensity and different RAPD profiling compared with wild type control. Four mutants were randomly selected and their extracellular enzyme activities were investigated. In liquid culture without casein, 2.2-, 2.9-, 5.2- and 4.4-fold higher acid protease activity was achieved from mutants DK-m9, -m11 and -m12, respectively, than that of wild type strain (11.13 ± 1.60 U/ml). In liquid culture with casein, 1.1-, 1.3-, 1.3 and 1.3-fold higher acid protease activity was achieved with those mutants were found to produce, respectively, than that of wild type strain (93.95 ± 12.84 U/ml). Maximum acid protease activity was noticed from a mutant DK-m11 in liquid culture with casein (121.18 U/ml) and without casein (57.65 U/ml). The extracellular acid protease produced from DJ-m11 that was active in the pH range 4.5-6.5 and optimum temperature for the activity was 37°C. Furthermore, we found a deformed, shorten structure of setae on the elytron surface of dynastid beetles treated with culture supernatant of the DK-m11. Conclusion : These findings have more impact on enzyme economy for biotechnological and insecticidal applications of fungal proteases.