In this study, we investigated the effects of pH, temperature, and salt concentration on protease activities in Hypsizygus marmoreus, and the ability of H. marmoreus extract to tenderize beef. In fact, H. marmoreus was shown to have higher protease activity than kiwi fruits. The pH of beef was dose dependently increased with addition of H. marmoreus extract. Cooking loss, cutting strength, and color value (L, a, b) were also dose dependently decreased with addition of H. marmoreus extract. In sensory assessments, H. marmoreus extract dose dependently enhanced perception of tenderness, flavor, and taste. In contrary, use of kiwi fruits as a meat tenderizer decreased positive perception of taste. The protease activitiy of H. marmoreus decreased sharply at pH < 2.0, and temperature > 50 oC. Protease activity was relatively stable in NaCl concentrations ranging from 0–5 M, but incrementally decreased with increasing NaCl. These results suggest that, H. marmoreus extract can improve the texture and taste of beef.