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Nitrogen is an important nutrient element that limits plant growth and yield formation, but excessive nitrogen has negative effects on plants and the environment. It is important to reveal the molecular mechanism of high NUE (nitrogen use efficiency) for breeding peach rootstock and variety with high NUE. In this study, two peach rootstocks, Shannong–1 (S) and Maotao (M), with different NUE were used as materials and treated with 0.1 mM KNO₃ for transcriptome sequencing together with the control group. From the results of comparison between groups, we found that the two rootstocks had different responses to KNO₃, and 2151 (KCL_S vs. KCL_M), 327 (KNO₃_S vs. KCL_S), 2200 (KNO₃_S vs. KNO₃_M) and 146 (KNO₃_M vs. KCL_M) differentially expressed genes (DEGs) were identified, respectively, which included multiple transcription factor families. These DEGs were enriched in many biological processes and signal transduction pathways, including nitrogen metabolism and plant hormone signal transduction. The function of <i>PpNRT2.1</i>, which showed up-regulated expression under KNO₃ treatment, was verified by heterologous expression in Arabidopsis. The plant height, SPAD (soil and plant analyzer development) of leaf and primary root length of the transgenic plants were increased compared with those of WT, indicating the roles of <i>PpNRT2.1</i> in nitrogen metabolism. The study uncovered for the first time the different molecular regulatory pathways involved in nitrogen metabolism between two peach rootstocks and provided gene reserve for studying the molecular mechanism of nitrogen metabolism and theoretical basis for screening peach rootstock or variety with high NUE.