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 NtNPF members showed significant genetic diversity within and across subfamilies but showed conservation between subfamilies. The NtNPF genes are dispersed unevenly across the chromosomes. The phylogenetic analysis revealed that eight subfamilies of NtNPF genes are tightly grouped with their orthologues in Arabidopsis. The promoter regions of the NtNPF genes had extensive cis-regulatory elements. Twelve core NtNPF 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 NtNPF genes and lay the foundation for further functional analysis of cigar tobacco.