To characterize the promoter region and upstream regulation region of human and mouse SCN3A gene, 5′-Full RACE was performed to identify that the nucleotide “A” was identified as the transcription start site, which locate 27 kb upstream of the translation start site of human SCN3A and 31 kb upstream of the translation start site of mouse SCN3A. Two 5′ -untranslated exons of human SCN3A and one 5′-untranslated exon of mouse SCN3A were found by sequence blast. The core promoter region (-80 ～ +70) of human SCN3A showed 96% nucleotide homology with that of mouse. Two core promoter elements, BRE and TATA, were predicted in the region of -80～+70 from both human and mouse. The transcriptional factors PHR1, GATA-1, FOXN2, NF-1 and AP-4 predicted in the region of -400 to +200 of human SCN3A, not found in mouse SCN3A, and the transcriptional factors Sp、Sp3 and GBF predicted in the region of -400 to +200 of mouse SCN3A, not found in human SCN3A. The comparison between the promoter region of human and mouse SCN3A may provide an important clue to explore the mechanisms of the regulations of human and mouse SCN3A expression.