The pharmacological actions of morphine and morphine-like drugs such as heroin mediate primarily through the mu opioid receptor (MOR). Previously, a single-strand DNA element of the mouse MOR proximal promoter was found to be important for regulating MOR gene in neuronal cells. To identify proteins binding to the single-strand DNA element as potential regulators for the MOR gene, affinity column chromatography with the single-strand DNA element was performed using the neuroblastoma NS20Y cells, followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified five poly(C)-binding proteins: hnRNP K, CP1, CP2, CP2-KL, and CP3. Binding of these proteins to the single-strand DNA element of the MOR gene was sequence-specific, as confirmed by supershift assays. In cotransfection studies, hnRNP K, CP1, CP2, and CP2-KL activated the MOR promoter activity, while CP3 acted as a repressor. Ectopic expression of hnRNP K, CP1, CP2, and CP2-KL also led to activation of the endogenous MOR transcripts and CP3 repressed endogenous MOR transcripts. We demonstrate novel roles as transcriptional regulators in MOR gene regulation for hnRNP K and CP binding to the single-strand DNA element.