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Tobacco prefers nitrate as a nitrogen (N) source. However, little is known about the molecular components responsible for nitrate uptake and the physiological responses of cigar tobacco to N deficiency. In this study, a total of 117 nitrate transporter 1 (NRT1) and peptide transporter (PTR) family (NPF) genes were comprehensively identified and systematically characterized in the whole tobacco genome. The <i>NtNPF</i> members showed significant genetic diversity within and across subfamilies but showed conservation between subfamilies. The <i>NtNPF</i> genes are dispersed unevenly across the chromosomes. The phylogenetic analysis revealed that eight subfamilies of <i>NtNPF</i> genes are tightly grouped with their orthologues in <i>Arabidopsis</i>. The promoter regions of the <i>NtNPF</i> genes had extensive <i>cis</i>-regulatory elements. Twelve core <i>NtNPF</i> genes, which were strongly induced by N limitation, were identified based on the RNA-seq data. Furthermore, N deprivation severely impaired plant growth of two cigar tobaccos, and CX26 may be more sensitive to N deficiency than CX14. Moreover, 12 hub genes respond differently to N deficiency between the two cultivars, indicating the vital roles in regulating N uptake and transport in cigar tobacco. The findings here contribute towards a better knowledge of the <i>NtNPF</i> genes and lay the foundation for further functional analysis of cigar tobacco.