Glutamine (Gln) is an efficient nitrogen source in promoting aboveground nitrogen and biomass accumulation in ZD958 (an elite maize hybrid with great potential for further genetic improvement) seedlings when conditioning a smaller but adequate root system. Amino acids account for a significant part of nitrogen (N) resources in the soil. However, how amino acid-N affects crop growth remains to be further investigated. Here, glutamine (Gln) application (80% NHNO + 20% Gln; mixed N) enhanced shoot growth of the maize hybrid ZD958. N concentration in the shoot increased, which is associated with favorable increases in SPAD values, GS/GOGAT activities, and accumulation of glutamate, asparagine, total free amino acids and soluble proteins in the shoot under mixed N. On the other hand, root growth was reduced when exposed to Gln as indicated by the significantly lower dry weight, root/shoot ratio, and primary, seminal, crown, and total root lengths, as well as unfavorable physiological alterations. Up-regulation of expression of ZmAMT1.3, ZmNRT2.1, and ZmAAP2 in the root and that of ZmAMT1.1, ZmAMT1.3, and ZmLHT1 in the shoot preconditioned N over-accumulation in the shoot and facilitated shoot growth, presumably via enhancing N translocation to the shoot, when Gln was supplied. Together, Gln is an efficient N source in promoting aboveground N and biomass accumulation in ZD958 seedlings when conditioning a smaller but adequate root system. Notably, ZD958's parental lines Z58 and Chang7-2 displayed a wide range of variations in Gln responses, which may be partially attributed to single nucleotide polymorphisms (SNPs) in cis-elements and coding regions revealed in this study and much larger quantities of unidentified genetic variations between Z58 and Chang7-2. Extensive genetic divergence of these two elite inbred lines implied large potentials for further genetic improvement of ZD958 in relation to organic N use efficiency.