Objective Voltage-gated sodium channel α subunit type Ⅰ (Nav1.1) is mainly expressed in the central nervous system.Abnormal expression of SCN1A (encoding Nav1.1) is associated with epilepsy.Two SCN1A promoters have been identified in human brain.The present study is to identify a third promoter of SCN1A and then to explore the functions of potential transcriptional elements in this promoter.Methods The minimal functional promoter and potential transcriptional elements were identified by 5 truncating or 3 extending analysis.The promoter activity was measured using the Dual-Luciferase Reporter Assay System (Promega).Transcription factor and DNA flagment binding assay in vitro was performed using Electrophoretic mobility shift assay (EMSA).Results (1) The third promoter fragment (P1c, nt-1023 to +52) showed a high activity in NGF-induced PC12 cells, but very low in SHSY5Y cells and HEK-293 cells.(2) The minimal functional promoter located within the region between nt-73 and nt +52.(3) The region nt +53 to nt +90 decreased the luciferase activity by ~75% that of the minimal functional promoter.(4) EMSA assay using a transcriptional repressor DREAM and the DNA fragment ranging from nt +53 to nt +90 showed that the DNA fragment was retarded after overexpression of DREAM in HEK293 cells.(5) Further truncating analysis demonstrated that the region between nt +53 and nt +62 was the DREAM binding site and was responsible for repressing the promoter activity.Conclusion The Ca2+ binding factor DREAM represses the third SCN1A promoter by binding the downstream repression element (DRE), which implicates a potential role of Ca2+ in the regulation of SCN1A expression in normal and diseased human tissues.