The proximal promoter of mouse mu-opioid receptor (MOR) gene is the dominant promoter for directing MOR-1 gene expression in brain. Sp1/Sp3 (Sps) and poly(C) binding protein 1 (PCBP) bind to a cis-element of MOR proximal promoter. Functional interaction between Sps and PCBP and their individual roles on MOR proximal core promoter were investigated using SL2 cells, devoid of Sps and PCBP. Each factor contributed differentially to the promoter, with a rank order of activity Sp1>Sp3>PCBP. Functional analysis suggested the interplay of Sps and PCBP in an additive manner. The in vivo binding of individual Sps or PCBP to MOR proximal promoter was demonstrated using chromatin immunoprecipitation (ChIP). Re-ChIP assays further suggested simultaneous bindings of Sps and PCBP to the proximal promoter, indicating physiologically relevant communication between Sps and PCBP. Collectively, results documented that a functional coordination between Sps and PCBP contributed to cell-specific MOR gene expression.