Abstract: The CLDN gene family encodes Claudins, the main transmembrane protein that constitutes tight junctions between cells and plays an important role in maintaining cell polarity, signal transduction, and recurrence and metastasis of malignant tumors. CLDN10 gene belongs to the CLDN family and has multiple transcripts including CLDN10 a. In this study, a variety of bioinformatics methods were used to analyze the characteristics of 5′ flanking sequence of CLDN10 a and transcription factor binding sites. Gene synthesis, PCR amplification and molecular cloning were used to construct CLDN10 a promoter luciferase reports of different lengths with 5′deletions. The gene vector was transiently transfected into HEK-293 T cells, and the promoter activity of different length fragments was detected by the dual luciferase reporter gene system. The results of bioinformatics analysis showed that the upstream transcription regulatory region sequence（-2 000～+460 bp） of CLDN10 a contained TATA box, GC box, CAAT box, 1 CpG island, 4 promoter positions, and 1 RNA polymeraseⅡcore promoter. And 75 transcription factor binding sites were predicted by MatInspector and Jaspar（Score≥0.99）. PCR and sequencing results showed that 6 dual luciferase reporter gene plasmids with diffe-rent lengths of CLDN10 a promoter were successfully constructed. The luciferase activity test showed that-1 890～-1 100 bp,-1 000～-890 bp,-890～-150 bp and-150～+460 bp were main active regions of human CLDN10 a promoter, while-150～+460 bp contained the core promoter region of CLDN10 a. This study provides experimental basis for further investigation of CLDN10 gene expression and regulation mechanism.