Background <p>NSUN6 (NOL1/NOP2/sun 6) is an mRNA m⁵C methylation-modified methyltransferase, whose aberrant expression or mutation leads to many cancers. There are fewer studies related to the regulatory mechanisms of NSUN6 itself.</p> Methods <p>A series of full-length and truncated plasmids of NSUN6 promoter region were constructed, and the core regulatory region of NSUN6 promoter was preliminarily characterized. Online website predicted the transcription factor binding sites in the core functional region, and the dual luciferase reporter gene assay, RT-qPCR, Western blot and ChIP assays confirmed the regulatory function of the transcription factor ATF3 on the NSUN6 promoter region.</p> Results <p>The NSUN6 promoter full-length and truncation plasmids were successfully constructed, and it was determined that the -74 to -34nt region of the NSUN6 promoter is the core region for regulating the transcriptional activity of the gene. ATF3 is the key transcription factor regulating the activity of the NSUN6 promoter. The transcriptional activity of NSUN6 was significantly decreased after mutating the ATF3 binding site. Overexpression and knockdown of ATF3 resulted in a concomitant increase and decrease in the transcriptional activity, mRNA and protein levels of the NSUN6. Chromatin immunoprecipitation assays confirmed that the transcription factor ATF3 could bind to the NSUN6 promoter region in vivo. Also, an expression correlation between NSUN6 and ATF3 was observed in several tumors.</p> Conclusion <p>The transcription factor ATF3 is a key transcription factor that positively regulates NSUN6 transcriptional activity. This provides new basic therapy and potential drug targets for NSUN6-related clinical diseases.</p>