A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds. Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.