Objective: To construct a luciferase reporter plasmid for human Sine oculis homeobox homolog 1(Six1) promoter fragment, predict the potential transcription factor binding sites, and to study the mechanism of E2F transcription factor 4(E2F4) on Six1. Methods: Six1 promoter fragment(-351~+100 nt) was synthesized and inserted into the luciferase reporter gene vector pGL3-basic, the activity of recombinant plasmid pSix1-451 in HEK-293 and BEAS-2B cells was measured by dual luciferase reporter gene assay, and the potential transcriptional binding sites were predicted by bioinformatics methods. Then pSix1-451 and siE2F4 or E2F4 overexpression plasmids were co-transfected into HEK-293 and BEAS-2B cells, the luciferase activity was measured to determine the role of E2F4 in Six1 gene transcription. The effects of knockdown and overexpression of E2F4 on Six1 gene expression were measured by qRT-PCR and Western blotting experiments. Results: The luciferase reporter plasmid of human Six1 promoter fragment was successfully constructed, and its activity was verified. The fragment contained transcription factor binding sites, such as E2F4. The regulatory effect of E2F4 on Six1 was verified. Conclusion: The transcription factor E2F4 could positively promote the expression of human Six1.