Beef steaks from longissimus lumborum, semimembranosus, and adductor muscles (n = 10; respectively) were cut at 24 h postmortem, randomly assigned to either high-oxygen modified atmosphere packaging (HiOx-MAP; 80% O2, 20% CO2) or vacuum (VAC), and displayed for 9 days at 1 °C. HiOx-MAP packaged beef steaks had a rapid increase in lipid oxidation and a decrease in color stability during display. The steaks in HiOx-MAP had significantly lower tenderness and juiciness scores, and higher off-flavor scores compared to steaks in VAC. HiOx-MAP condition did not affect the postmortem degradation of troponin-T or desmin. Furthermore, autolysis of μ-calpain was not influenced by packaging. SDS–PAGE, immunoblotting, and diagonal-PAGE revealed oxidative cross-linking of myosin heavy chain in meat packaged in HiOx-MAP. These results suggest that the HiOx-MAP system may negatively affect meat quality characteristics by inducing lipid and myoglobin oxidation and cross-linking/aggregation of myosin by protein oxidation.