Sucrose (Suc) is directly associated with plant growth and development as well as tolerance to various stresses. Invertase (INV) enzymes played important role in sucrose metabolism by irreversibly catalyzing Suc degradation. However, genome-wide identification and function of individual members of the INV gene family inNicotiana tabacumhave not been conducted. In this report, 36 non-redundant NtINV family members were identified inNicotiana tabacumincluding 20 alkaline/neutral INV genes (NtNINV1-20), 4 vacuolar INV genes (NtVINV1-4), and 12 cell wall INV isoforms (NtCWINV1-12). A comprehensive analysis based on the biochemical characteristics, the exon-intron structures, the chromosomal location and the evolutionary analysis revealed the conservation and the divergence of NtINVs. For the evolution of the NtINV gene, fragment duplication and purification selection were major factors. Besides, our analysis revealed that NtINV could be regulated by miRNAs and cis-regulatory elements of transcription factors associated with multiple stress responses. In addition, 3D structure analysis has provided evidence for the differentiation between the NINV and VINV. The expression patterns in diverse tissues and under various stresses were investigated, and qRT-PCR experiments were conducted to confirm the expression patterns. Results revealed that changes inNtNINV10expression level were induced by leaf development, drought and salinity stresses. Further examination revealed that the NtNINV10-GFP fusion protein was located in the cell membrane. Furthermore, inhibition of the expression ofNtNINV10gene decreased the glucose and fructose in tobacco leaves. Overall, we have identified possibleNtINVgenes functioned in leaf development and tolerance to environmental stresses in tobacco. These findings provide a better understanding of theNtINVgene family and establish the basis for future research.