Abstract: Expansins play important roles in plant growth and development and plant's responses to abiotic stresses. Cloning and studying the functions of tobacco expansin genes will provide a theoretical basis for regulation of tobacco leaf development and tobacco resistance breeding. In this study, we cloned NtabEXPA12 with its promoter ProNtabEXPA12 from tobacco variety K326, studied its function by qPCR, subcellular localization and gene overexpression assays, and analyzed cis-regulatory elements in ProNtabEXPA12 and its promoter activity. The results showed that NtabEXPA12 was highly expressed in tobacco leaves. Various cis-elements in ProNtabEXPA12 were identified which were related to plant's responses to salt, drought and plant hormones. And indeed, the activity of ProNtabEXPA12 was strongly induced by NaCl, ABA and drought treatments. We also found that overexpression of NtabEXPA12 promoted cell and leaf expansion, and enhanced abiotic stress tolerance in transgenic K326 plants. Therefore, NtabEXPA12 has potential application values in manipulating tobacco leaf development and tobacco resistance breeding.