Ethylenediurea (EDU) has been used as a chemical protectant against ozone (O). However, its protective effect and physiological mechanisms are still uncertain. The present study aimed to investigate the changes of foliar visible injury, physiological characteristics and emission rates of volatile organic compounds (VOCs) in one-year-old Populus alba "Berolinensis" saplings pretreated with EDU and exposed to elevated O (EO, 120 μg/m). The results showed that foliar visible injury symptoms under EO were significantly alleviated in plants with EDU application (p < 0.05). Under EO, net photosynthetic rate, the maximum photochemical efficiency of PSII and the photochemical efficiency of PSII of plants pretreated with 300 and 600 mg/L EDU were similar to unexposed controls and significantly higher compared to EO-stressed plants without EDU pretreatment, respectively. Malondialdehyde content was highest in EO without EDU and decreased significantly by 14.9% and 21.3% with 300 and 600 mg/L EDU pretreatment, respectively. EDU pretreatment alone increased superoxide dismutase activity by 10-fold in unexposed plants with further increases of 88.4% and 37.5% in EO plants pretreated with 300 and 600 mg/L EDU pretreatment, respectively (p < 0.05). Abscisic acid content declined under EO relative to unexposed controls with the effect partially reversed by EDU pretreatments. Similarly, VOCs emission rate declined under EO relative to unexposed plants with a recovery of emission rate observed with 300 and 600 mg/L EDU pretreatment. These findings provided significant evidence that EDU exerted a beneficial effect and protection on the tested plants against O stress.