Elucidating physiological mechanisms underlying the plant N uptake benefits breeding of high N use efficiency (NUE) crop cultivars. In this study, we investigated the growth and N uptake-associated processes in wheat under N deprivation and deficit irrigation, using two contrasting NUE cultivars. Compared with sufficient-N (SN), deficient-N (DN) treatment reduced plant biomass, N accumulation, and yields in two cultivars (high NUE Shinong 086 and N deprivation-sensitive Jimai 585), suggesting that N deprivation negatively regulates plant growth and N uptake. Shinong 086 was better on growth and N uptake-associated traits than Jimai 585 due to the improved root biomass across soil profile, which was consistent with the decrease of available N contents in soil layers. These results suggested that the improved root system architecture (RAS) enhances plant acquirement for soil N under N- and water-deprivation condition, contributing to the plant N uptake and yield formation capacities. Transcriptome investigation revealed that numerous genes were differentially expressed (DE) in the N-deprived Shinong 086 plants, which involve the regulation of complicate biochemical pathways. These results suggested that the modified RAS and N uptake in high NUE plants are accomplished underlying the regulation of numerous DE genes. TaWRKY20, a gene in ZFP transcription factor family, was functionally characterized for the role in mediating plant N uptake. Overexpression of it conferred plants improved growth and N uptake under DN due to its regulation on TaNRT2.1 and TaNRT2.2, two nitrate transporter genes. Our investigation provides insights in high NUE mechanisms in wheat under N deprivation.