Abstract: The process of nicotine conversion, particularly the synthesis of nornicotine from nicotine during the mature stage of tobacco leaves, significantly impacts the quality of tobacco leaves. Previous research has identified that this conversion is facilitated by nicotine N-demethylase, which is encoded by the gene CYP82E4 (NtE4). To understand the regulatory mechanisms and function of the NtE4 promoter, this study identified cis-acting elements on the NtE4 promoter and predicted potential upstream transcription factors by analyzing online databases. Through the deletion of the 5' end, recombinant expression vectors of promoter fusion GUS gene were constructed to transform Nicotiana benthamiana transiently, and the active region of the promoter was determined. The findings revealed that the NtE4 promoter contains 2 abscisic acid and 11 ethylene response elements, as well as 9 and 7 binding sites for WRKY and NAC transcription factors respectively. The core region of the NtE4 promoter was identified to be between −500 and −1000 bp, with the region spanning from −500 to −1967 bp enhancing promoter activity. Furthermore, upstream transcription factors such as ERF, WRKY, and NAC were predicted to regulate the NtE4 gene. Fluorescence quantitative expression analysis demonstrated that ERF5, WRKY50, and NAC29 exhibited similar expression patterns to NtE4 during leaf senescence. This study contributes to understanding the regulatory mechanisms underlying NtE4-mediated nicotine conversion during tobacco aging, providing a foundation for further research into this process.