Although biological functions of ammonium (NH) transporters (AMTs) have been intensively studied in many plant species, little is known about molecular bases responsible for NH movement in tobacco. Here, we reported the identification and functional characterization of a putative NH transporter NtAMT1.3 from tobacco (Nicotiana tabacum). Analysis in silico showed that NtAMT1.3 encoded an integral membrane protein containing 464 amino acid residues and exhibiting 10 predicted transmembrane α-helices. Heterologous functionality study demonstrated that NtAMT1.3 expression facilitated NH entry across plasma membrane of NH-uptake defective yeast and Arabidopsis qko mutant, allowing a restored growth of both yeast and Arabidopsis mutant on low NH. qPCR assay revealed that NtAMT1.3 was expressed in both roots and leaves and significantly up-regulated by nitrogen starvation and resupply of its putative substrate NH and even nitrate, suggesting that NtAMT1.3 should represent a nitrogen-responsive gene. Critically, constitutive overexpression of NtAMT1.3 in tobacco per se improved obviously the growth of transgenic plants on NH and enhanced leaf nitrogen (15% more) accumulation, consistent with observation of 35% more NH uptake by the roots of transgenic lines in 20min root-influx test. Together with data showing its plasma membrane localization and saturated transport nature with K of about 50μM for NH, we suggest that NtAMT1.3 acts an active NH transporter that plays a significant role in NH acquisition and utilization in tobacco.