‘Danhong’ (Populus deltoides) and ‘Tongliao 1’ (Populus simonii) were used as materials, and treatments with and without nitrogen fertilization were conducted in the field. The changes in growth traits, carbon, and nitrogen-related metabolites, and transcriptome of the developing xylem were analyzed. The physiological mechanism of nitrogen fertilizer utilization in different poplar varieties was discussed, which laid a foundation for the genetic breeding of nitrogen efficient utilization of poplar. The results showed that: (1) The total biomass of ‘Danhong’ and ‘Tongliao1’ poplar increased 1.69 times and 1.10 times, respectively, under nitrogen application treatment. The total biomass of ‘Danhong’ poplar was 13 times higher than that of ‘Tongliao 1’ poplar under the condition of nitrogen fertilization and 10 times higher than that of ‘Tongliao 1’ poplar under the condition of no nitrogen fertilization. (2) The treatment of nitrogen fertilizer significantly inhibited the contents of total nitrogen and various hydrolyzed amino acids in the bark and xylem of ‘Danhong’ and ‘Tongliao 1’, but did not significantly affect the contents of total carbon, cellulose, hemicellulose, and lignin in the xylem. (3) Nitrogen fertilizer treatment significantly affected the high expression of genes in carbon and nitrogen metabolic pathways such as carbon fixation, sugar metabolism, and amino acid synthesis in the developing xylem of two poplar species, which resulted in biomass accumulation. In this study, we found that nitrogen application treatment significantly promoted the high expression of genes related to the carbon and nitrogen metabolic pathways in the developing xylem of poplar, thus promoting the accumulation of biomass and growth of poplar. The wood yield of ‘Danhong’ poplar was much higher than that of ‘Tongliao 1’ poplar under different nitrogen environments, which was more suitable for large-scale promotion and planting of plantation.