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To investigate the effect of nitrogen (N) level on iron (Fe) content in milled rice, a field experiment was carried out under three N application levels including 0, 150 and 300 kg/hm2 by using 120 rice genotypes. In addition to the genotypic differences of iron content in milled rice, grain yield, 1000-grain weight and N content in grains under the same N level, there were also variations in the response of Fe content in milled rice to N levels. Based on the range and variation coefficient of Fe content in milled rice under the three N levels, the response of Fe content in milled rice to N levels could be classified into four types including highly insensitive, insensitive, sensitive and highly sensitive types. A significant quadratic correlation was found between the Fe content in milled rice and 1000-grain weight or the N content in grains. However, no significant correlation between the Fe content in milled rice and grain yield was detected. In conclusion, there are genotypic differences in the effects of N levels on Fe content in milled rice, which is favorable to breeding of Fe-rich rice under different N environments. Furthermore, high yield and Fe-rich rice could be grown through the regulation of nitrogen on Fe content in milled rice, 1000-grain weight and N content in milled rice.