Abstract: Nornicotine is a harmful alkaloid, and its accumulation in tobacco is associated with leaf maturity and senescence. To reduce nornicotine in tobacco leaves, we explored the regulation of senescence-induced nornicotine synthesis. Based on our previous preliminary finding that NtWRKY51 is induced by senescence, we cloned the CDS sequence of NtWRKY51and conducted analyses including bioinformatics characterization, spatiotemporal expression pattern, subcellular localization, gene function, and identification of downstream interacting gene. The results showed that the full-length CDS of NtWRKY51 is 576 bp, encoding a protein of 191 amino acids, and that it belongs to the WRKYII subfamily. NtWRKY51 is a nuclear localized protein, which is highly expressed in tobacco leaves and stems, with its expression induced by senescence. The nornicotine content in senescent leaves of NtWRKY51-CR lines is significantly increased, reaching more than 1.6-fold that of WT in fully senescent leaves. Conversely, overexpression of NtWRKY51 significantly reduces nornicotine content by up to 40%. These results indicates that NtWRKY51 negatively regulates nornicotine biosynthesis during senescent leaves. Further studies showed that NtWRKY51 binds to the promoter region of the key nornicotine biosynthetic enzyme gene CYP82E4 and inhibits its transcription. The relative expression of CYP82E4 in knockout lines is significantly higher than in WT, whereas its expression in overexpression lines is significantly lower than in WT. Taken together, although NtWRKY51 is induced by senescence, it plays a negative regulatory role in the senescence-induced nornicotine synthesis process by inhibiting CYP82E4 expression.