A sufficient nitrogen (N) supply is pivotal for high grain yield and desired grain protein content in wheat (Triticum aestivum L.). Elucidation of physiological and molecular mechanisms underlying nitrogen use efficiency (NUE) will enhance our ability to develop new N-saving varieties in wheat. In this study, we analyzed two soft red winter wheat genotypes, VA08MAS-369 and VA07W-415, with contrasting NUE under limited N. Our previous study demonstrated that higher NUE in VA08MAS-369 resulted from accelerated senescence and N remobilization in flag leaves at low N. The present study revealed that VA08MAS-369 also exhibited higher nitrogen uptake efficiency (NUpE) than VA07W-415 under limited N. VA08MAS-369 consistently maintained root growth parameters such as maximum root depth, total root diameter, total root surface area, and total root volume under N limitation, relative to VA07W-415. Our time-course N content analysis indicated that VA08MAS-369 absorbed N more abundantly than VA07W-415 after the anthesis stage at low N. More efficient N uptake in VA08MAS-369 was associated with the increased expression of genes encoding a two-component high-affinity nitrate transport system, including four NRT2s and three NAR2s, in roots at low N. Altogether, these results demonstrate that VA08MAS-369 can absorb N efficiently even under limited N due to maintained root development and increased function of N uptake. The ability of VA08MAS-369 in N remobilization and uptake suggests that this genotype could be a valuable genetic material for the improvement of NUE in soft red winter wheat.