In this research, the effect of high-oxygen packaging (HOP) with initial 80% and 100% oxygen on fresh shiitake mushrooms was studied. Initial air in package was the control treatment. All the samples were stored at 10 °C with RH 90% for 9 days. Respiration rate, hardness, TSS, and color were determined, and sensory quality was evaluated during the storage. Results indicated that high-oxygen packaging retarded the anaerobic metabolism occurrence and HOP with initial 100% oxygen could maintain the lightness of shitake mushroom better than 80% oxygen. Hardness and TSS did not show significant difference between high-oxygen packaging and control treatment. However, neither initial 100% O₂ nor 80% O₂ could reduce the respiration rate of fresh shiitake mushroom. Sensory quality especially the aroma, cap color and gill color of fresh shiitake mushroom could be better maintained in high-oxygen packaging than control treatment. The acceptability of the shiitake mushrooms after storage was the highest in HOP with initial 100% oxygen. In conclusion, high-oxygen packaging especially with initial 100% oxygen showed the obvious effect maintaining the sensory quality of fresh shiitake mushrooms although it could not reduce the respiration rate at 10 °C.