To further study the molecular mechanism of transcription regulation of the mouse(Mus musculus)histone H3K4 methylase gene Smyd3, different 5'-flanking regions of Smyd3 promoter were cloned by PCR and inserted into pMD19-T vector. The pMD19-T vector was digested by two enzymes, and then inserted into pGL3-Basic luciferase reporter vector. The recombination plasmids were transiently transfected into HEK293 cells, and then its fluorescence activity was measured with the dual-luciferase reporter assay after 48 hours. The results showed that pGL3-Basic-Smyd3-1-pGL3-Basic-Smyd3-5 luciferase reporter gene vectors were constructed successfully. Compared with the positive control, construction of promoter recombinants transfected group showed fluorescence activity, and the pGL3-Smyd3-4 fluorescence was most active, about 2-4 times of the others, while the pGL3-Smyd3-5 fluorescence activity was the weakest. This study suggests that the core promoter region of the Smyd3 gene may be located on the up-stream between -533 bp to -42 bp and that the area between -2 026 bp to -533 bp is a transcriptional negative regulation region.