The ammonium transporter (AMT) family gene is an important transporter involved in ammonium uptake and transfer in plants and is mainly engaged in the uptake and transport of ammonium from the environment by roots and the reabsorption of ammonium in the aboveground parts. In this study, the expression pattern, functional identification, and genetic transformation of thePtrAMT1;6gene, a member of the ammonium transporter protein family inP. trichocarpa, were investigated as follows: (1) Fluorescence quantitative PCR demonstrated that thePtrAMT1;6gene was preferentially expressed in the leaves, with both dark-induced and light-inhibited expression patterns. (2) A functional restoration assay using the yeast ammonium transporter protein mutant strain indicated that thePtrAMT1;6gene restored the ability of the mutant to transport ammonium with high affinity. (3)Arabidopsiswas transformed with pCAMBIA-PtrAMT1;6P,and the transformed lines were stained with GUS, which showed that the rootstock junction, cotyledon petioles, and the leaf veins and pulp near the petioles of the transformed plants could be stained blue, indicating that the promoter of thePtrAMT1;6gene had promoter activity. (4) The overexpression of thePtrAMT1;6gene caused an imbalance in carbon and nitrogen metabolism and reduced nitrogen assimilation ability in ‘84K’ poplar and ultimately reduced biomass. The above results suggest thatPtrAMT1;6may be involved in ammonia recycling during nitrogen metabolism in aboveground parts, and overexpression ofPtrAMT1;6may affect the process of carbon and nitrogen metabolism, as well as nitrogen assimilation in plants, resulting in stunted growth of overexpression plants.