Improving nitrogen use efficiency (NUE) is very important for crops throughout the world. Rice mainly utilizes ammonium as an N source, but it also has four NRT2 genes involved in nitrate transport. The OsNRT2.3b transporter is important for maintaining cellular pH under mixed N supplies. Overexpression of this transporter driven by a ubiquitin promoter in rice greatly improved yield and NUE. This strategy for improving the NUE of crops may also be important for other cereals such as wheat and barley, which also face the challenges of nutrient uptake balance. To test this idea, we constructed transgenic barley lines overexpressing OsNRT2.3b. These transgenic barley lines overexpressing the rice transporter exhibited improved growth, yield, and NUE. We demonstrated that NRT2 family members and the partner protein HvNAR2.3 were also up-regulated by nitrate treatment (0.2 mM) in the transgenic lines. This suggests that the expression of OsNRT2.3b and other HvNRT2 family members were all up-regulated in the transgenic barley to increase the efficiency of N uptake and usage. We also compared the ubiquitin (Ubi) and a phloem-specific (RSs1) promoter-driven expression of OsNRT2.3b. The Ubi promoter failed to improve nutrient uptake balance, whereas the RSs1 promoter succeed in increasing the N, P, and Fe uptake balance. The nutrient uptake enhancement did not include Mn and Mg. Surprisingly, we found that the choice of promoter influenced the barley phenotype, not only increasing NUE and grain yield, but also improving nutrient uptake balance.