Adventitious roots (ARs) formation is crucial for the vegetative propagation of horticultural crops. Nitrate availability is the main factor determining plant growth and development. Nitrate uptake and distribution is carried out by nitrate transporters (NRTs). In present work, we identified 84 MdNRT genes in the apple genome and analyzed them in terms of phylogenetic analysis, syntenic relationships, domain structure, cis-elements, and motif analysis. We further analyzed their amino acid composition, physical and chemical characteristics. Our results revealed that apple NRT family can be divided into three subfamilies; MdNRT1/PTR, MdNRT2, and MdNRT3 having 77, 5, and 2 members, respectively. The syntenic relationships indicated that almost 48.8 % of the total NRT genes appeared from genome duplication. The composition of cis-elements suggests that most of the MdNRT genes might be involved in hormones, light, and stress responses. Moreover, different nitrate treatments (9.4 mM/L, 18.8 mM/L, 28.1 mM/L, 46.9 mM/L, and 84.5 mM/L) were applied, and 28.1 mM/L was identified as an optimum level of nitrate for ARs formation and development in B9 apple rootstock. Additionally, RNA sequencing results revealed that several MdNRT genes showed an organ-specific expression pattern suggesting their role in nitrate distribution. These MdNRT genes indicated diverse expression profiles in response to nitrate treatments (28.1 mM/Land 46.9 mM/L) in the stem basal parts of B9 apple rootstock during AR formation. Interestingly, MdNRT2.1 gene has the strongest response to nitrate availability, advocating that MdNRT2.1 may have a significant role in the apple ARs formation and development. Furthermore, over-expression of 35 s MdNRT2.1 in tobacco increased root number (53.6 %), length (72.5 %), volume (54.6 %), and surface area (58.4 %) compared with wild type. Therefore, we concluded that MdNRT2.1 has a direct role in AR formation and development. Our research provides new genetic resources for functional characterization of NRT genes.