Abstract: To clarify the function of non-specific lipid transfer protein gene NtLTP1.1 in resistance against disease and stress, full-length NtLTP1.1 was cloned from Nicotiana tabacum by RACE technology, and cDNA sequence structure, protein physicochemical properties, conserved amino acid motifs, tertiary structure and evolutionary relationships were analyzed with bioinformatics methods. The expression patterns in different tissues and expression difference under stress of NtLTP1.1 were determined by qRT-PCR. The results showed that NtLTP1.1 was 615 bp in length and encoded a protein of 129 amino acids, the protein was a micromolecular soluble basic protein, and its N-terminal contained a signal peptide that might target the protein in secretory pathway. Blast analysis revealed that the homology of sequence was the highest between tobacco NtLTP1.1 and tomato nsLTP1, NtLTP1.1 contained a conserved 8CM motif composed of eight-cysteine-motif. The prediction analysis of protein tertiary structure indicated that NtLTP1.1 possessed the typical tertiary structure of nsLTP, which was composed of 4 α-helices, 4 pairs of disulphide bonds, an internal hydrophobic cavity capable of binding and accommodating lipid molecule. The multiple alignment results showed the structural domain pattern of NtLTP1.1 8CM was C1-X9-C2-X13-C3C4-X19-C5XC6-X22-C7-X13-C8. Phylogenetic analysis revealed that NtLTP1.1 belonged to the Type I among the 5 clustered types of nsLTP. The results of qRT-PCR analysis showed that NtLTP1.1 was a tissue-specific gene and mainly expressed in tobacco leaves, its expression was suppressed under low temperature stress, while promoted by salicylic acid induction; it suggested that NtLTP1.1 gene might play a role in tobacco defense reaction.