Abstract: WRKY transcription factor family is plant-specific and WRKY53, a member of this gene family, plays a significant role in plant biotic and abiotic stress response and leaf senescence. To explore the biological functions of NtWRKY53 gene in Nicotiana tabacum, the NtWRKY53 gene of tobacco was cloned. WRKY transcription factor encoded by NtWRKY53 gene was proved to possess a highly conservative and typical WRKY domain by domain analysis. Phylogenetic and collinearity analyses revealed that NtWRKY53 gene of Nicotiana tabacum and AtWRKY53 gene of Arabidopsis were paralogous genes. Subcellular localization assay indicated that NtWRKY53 transcription factor was localized in cell nucleus. Transactivation analysis implied that NtWRKY53 transcription factor displayed transcriptional activation activity. In addition, the results of expression pattern analysis showed that the NtWRKY53 gene was up-regulated in the senescent leaves and could be induced by multiple stress treatments, such as salt and drought stresses, CMV and black shank disease. Therefore, NtWRKY53 gene encoded a WRKY transcription factor while localized in the nucleus and had a high expression level in yellowing tobacco leaves. It was induced by biotic and abiotic stresses, thus taking part in the biotic and abiotic stress responses.