In this study, we determined the capacity of soybean (Glycine max L. Merr. cv. Hoyoharuka) and sorghum (Sorghum bicolor L. Moench. cv. Hybrid Sorgo) to utilize different forms of nitrogen (N) in a rhizobox system. Seedlings were grown for 35 days without N or with 130 mg N kg-1 soil as ammonium sulfate or farmyard cattle manure. The soil fractions at different distances from the root were sliced millimeter by millimeter in the rhizobox system. We assessed the distribution of different forms of N and microbial metabolism in different soil fractions in the rhizosphere. There are no treatment-dependent changes in biomass production in the roots and shoots of soybeans, however, the ammonium and manure treatment yielded 1.30 and 1.40 times higher shoot biomass of sorghum than the control. Moreover, the depletion of inorganic N and total amino acids (TAA) in the rhizosphere was largely undetectable at various distances from the soybean roots regardless of the treatments employed. The addition of ammonium sulfate resulted in a decrease in the nitrate concentration gradient as the distance decreased from the sorghum roots. The addition of manure to the soil increased the N content in the sorghum shoots, 1.57 times higher than the control; this increase was negatively correlated with the concentrations of TAA in the soil of the root compartment. In addition, the application of manure simultaneously induced TAA depletion (i.e., the TAA concentration in root compartment was 1.48 times higher than that in bulk soil) and greater microbial activity and diversity in the sorghum rhizosphere, where higher microbial consumption of asparagine, glutamic acid, and phenylalanine were also observed near the roots. Our results are first to present the evidence that sorghum may possess a high capacity for taking up amino acids as a consequence of organic matter application, and microbial metabolism.