Abstract: Using Lonicera japonica genomic DNA as a template, we cloned Lonicera japonica U6 promoters. Four LjU6 promoters, 336, 708, 359 and 602 bp in length, were cloned by PCR from Lonicera japonica genomic DNA. PlantCARE analysis found that the four promoters contained typical promoter cis-elements, such as a TATA box and CAAT box, and contained regulatory elements related to light response and stress response. After the cloning products were sequenced, the LjU6 promoter was ligated to the pBI121 vector carrying the β-glucuronidase (GUS) gene to construct four LjU6-pBI121 fusion expression vectors. Nicotiana tabacum leaves were transformed by the Agrobacterium transient transformation method and GUS histochemical staining was performed on the leaves. The staining results showed that LjU61-F1 had the highest transcriptional activity. This study thus identified a U6 promoter with high transcriptional activity, providing a basis for the establishment of CRISPR/Cas9 genome editing technology in Lonicera japonica.