Abstract: In this study, we choose wheat cultivars “Shi 4185” and “Cang 6001” as materials to examine effects of antioxidative levels on iron deficiency tolerance and iron accumulation in wheat (Triticum aestivum L.) seedlings. The results show that iron deficiency markedly increases reactive oxygen species (ROS) level in roots, thereby induces oxidative damage in roots of wheat seedlings and reduces the chlorophyll content in leaves. Compared to “Shi 4185”, “Cang 6001” seedlings have lower ROS level, higher chlorophyll content, more lateral roots and higher leaves iron accumulation, as well as higher SOD, CAT and APX activities as exposed to iron deficiency. Supplementation of antioxidants such as ascorbic acid (AsA) and N-acetyl-L-cysteine (NAC) increases Fe content in wheat seedlings, suggesting higher antioxidative level improves Fe absorption and accumulation in wheat seedlings. This study has positive significance for further understanding the physiological mechanisms of wheat in response to iron deficiency stress and screening wheat cultivars for high iron deficiency tolerance and iron accumulation.