Abstract： Objective To investigate the role of Sp3 in the transcriptional regulation of enamelin gene. Methods By bioinformatic analysis, a putative responsive element for Sp3 was identified.Electrophoretic mobility shift assay was used to examine the interaction between Sp3 and enamelin.5'-flanking regulatory region of enamelin was cloned and ligated into pGL3-basic luciferase vector. Sp3 and the Enam-luc were cotransfected into mouse ameloblast-like cell line, and the activity of luciferase was examined. Results The results showed that Sp3 could not directly bind to the enamelin regulation region and activate enamelin transcription. Conclusions Sp3 might not be involved in transcriptional regulation of enamelin gene via an indirect interaction.