The concentration of ozone (O₃) is believed to continually increase due to anthrogenic activity, which might affect severely the growth and grain quality of winter wheat (Triticum aestivum). Flixweed (Descurainia sophia) is one of the most troublesome annual dicot weeds in wheat fields. As a vigourous competitor, the presence of flixweed in wheat field is a major factor to reduce wheat production. However, few studies have investigated the effects of ozone on wheat growth and yield under competition with flixweed. In the present study, physiological and ecological responses of wheat to O₃ stress with different density of flixweed competition were reported. Chlorophyll concentration and leaf area were reduced by elevated O₃. Gas exchange of wheat was suppressed by both elevated O₃ and the presence of flixweed. Moreover, exposure of wheat to elevated O₃ induced accumulation of Proline due to the up-regulation of pyrroline-5-carboxylate synthetase at transcriptional level, suggesting that Proline may confer tolerance to oxidative stress induced by O₃ pollution. Finally, elevated O₃ had significant adverse impacts on wheat above-ground biomass, grain yield and harvest index, and these impacts were intensified by the presence of flixweed. Exposure of wheat to elevated O₃ led to a significant increase in crude protein concentration of grain, while decreases in mineral element concentrations of grain were observed. Our findings indicate that effects of ozone and flixweed competition on winter wheat should be considered for the future breeding and cultivation.