Abstract: To study the function of Nt14-3-3 gene family in tobacco (Nicotiana tabacum), Nt14-3-3 was cloned from cv. K326 by homologous cloning. In addition, bioinformatics analysis of Nt14-3-3 was carried out. The transcriptional expressions of Nt14-3-3 were analyzed by qRT-PCR in different tissues of tobacco seedlings under abiotic stresses. The results showed that the gene sequence contained 783 bp in length, and encoded 260 amino acid residues. Nt14-3-3 was a globular protein contained nine highly conserved α helixes and 32 phosphorylation sites. Tissue expression analysis showed that Nt14-3-3 was expressed in tobacco roots, stems, leaves and flowers, especially in roots. The expression levels of Nt14-3-3 were induced under abiotic stress treatments (low potassium, high salinity, drought, ABA and H₂O₂), which indicated that Nt14-3-3 was involved in tobacco abiotic stress response.