AbstractBACKGROUNDOzone (O3), a widespread air pollutant, significantly impairs crop growth and development, with wheat, the second largest crop by planting area of the world, being especially vulnerable. This study, conducted under Free Air Concentration Enrichment (FACE) conditions, focused on four wheat cultivars from the middle and lower reaches of the Yangtze River, investigating the effects of elevated O3 on wheat growth, physiology and quality.RESULTSElevated O3 levels impaired assimilate accumulation and mobilization in wheat grains, reducing pre‐anthesis nitrogen accumulation and causing an 8.21% decline in post‐anthesis nitrogen translocation amount (NT), while increasing post‐anthesis nitrogen translocation efficiency (NTE) by 3.83% and nitrogen harvest index (NHI) by 3.43%. Over 2 years, elevated O3 raised grain protein content by 6.6–6.7% but significantly reduced protein accumulation by 10.4–10.7%, driven by declines in gliadin and glutenin. Total free amino acids and key nitrogen metabolizing enzymes also decreased. Among the four cultivars, YN19 was the most sensitive, showing the largest protein accumulation reductions.CONCLUSIONThis study demonstrates that elevated O3 disrupts wheat nitrogen accumulation and protein synthesis by reducing pre‐anthesis nitrogen accumulation, accelerating post‐anthesis senescence, and suppressing nitrogen translocation due to rapid leaf area index (LAI) decline. The decline in nitrogen accumulation and nitrogen metabolizing enzyme activity is a critical factor contributing to reduced grain protein accumulation. Notably, YN19 exhibited the highest O3 sensitivity, underscoring the need to develop O3‐resilient wheat cultivars to sustain grain quality under rising O3 levels. © 2025 Society of Chemical Industry.