As an abundant ROS, hydrogen peroxide (H2O2) plays pivotal roles in plant growth and development. In this work, we conducted for the first time an iTRAQ-based quantitative proteomic analysis of wheat seedling growth under different exogenous H2O2 treatments. The growth of seedlings and roots was significantly restrained by increased H2O2 concentration stress. Malondialdehyde, soluble sugar, and proline contents as well as peroxidase activity increased with increasing H2O2 levels. A total of 3 425 proteins were identified by iTRAQ, of which 157 showed differential expression and 44 were newly identified H2O2-responsive proteins. H2O2-responsive proteins were mainly involved in stress/defense/detoxification, signal transduction, and carbohydrate metabolism. It is clear that up-regulated expression of signal transduction and stress/defence/detoxification-related proteins under H2O2 stress, such as plasma membrane intrinsic protein 1, fasciclin-like arabinogalactan protein, and superoxide dismutase, could contribute to H2O2 tolerance of wheat seedlings. Increased gluconeogenesis (phosphoenol-pyruvate carboxykinase) and decreased pyruvate kinase proteins are potentially related to the higher H2O2 tolerance of wheat seedlings. A metabolic pathway of wheat seedling growth under H2O2 stress is presented.