The 5′-flanking region of the mouse μ opioid receptor (MOR) gene has two promoters, referred to as distal and proximal. MOR mRNA is predominantly initiated by the proximal promoter. Previously, several important cis-elements and transfactors have been shown to play a functional role in the proximal promoter of the MOR gene. In this study, we defined another functional, negative regulatory element located in the -219- to -189-base pair (translational start site designed as + 1) region of the proximal promoter. It is designated as the Sp binding sequence for its sequence homology to the consensus Sp binding element. Mutation of the Sp binding element led to a 100% increase of MOR promoter activity in MOR-positive cells (NMB cells), confirming the negative role of the Sp binding sequence. Surprisingly, electrophoretic mobility shift analysis and chromatin immunoprecipitation assays revealed that Sp3 and its isoforms (M1 and M2) were specifically bound to the Sp binding sequence. In cotransfection assays of Drosophila melanogaster SL2 cells using cDNA encoding Sp1, Sp3, and the M1 and M2 isoforms of Sp3, the M1 and M2 isoforms trans-repressed the MOR promoter, whereas Sp1 and Sp3 trans-activated the MOR promoter. Significantly, ectopic expression of the M1 and M2 isoforms of Sp3 led to repression of the endogenous MOR gene transcripts in NMB cells. These results suggest that the binding of the M1 and M2 isoforms of the Sp3 transcription factor to the Sp binding sequence may play a role in mouse MOR gene expression.