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Sunflower seeds are rich in oil and protein. In a two-year field experiment in Hetao district, Inner Mongolia, we evaluated the effects of irrigation and N fertilizer on protein and amino acid content of sunflower seeds (Kang Di T562 variety). Irrigation water and N fertilizer were applied at the bud to blossom stage and at three levels: water at 90, 45, and 0 mm; N fertilizer at 104, 52, and 0 kg hm−2. There were nine treatments with three replicates for a total of 27 plots arranged randomly in blocks. In combination with environmental conditions, water, nitrogen, and the interaction between the two had significant effects on total protein, total amino acids, total ammonia, and key amino acids: glutamic acid, arginine, aspartic acid, glycine, and valine. Application of N fertilizer at the bud stage promoted protein synthesis in seed kernels at the ripening stage. We found a significant positive correlation between N fertilizer and the five main amino acids. With increasing irrigation, total amino acid content and the content of each of the key amino acids individually first increased but then decreased. Overall, a water deficit improved protein content in seed kernels. [ABSTRACT FROM AUTHOR]