Understanding the regulation of a heterologously expressed gene cluster in a host organism is crucial for activation of silent gene clusters or overproduction of the corresponding natural product. In this study, Streptomyces coelicolor M512(nov-BG1) containing the novobiocin biosynthetic gene cluster from Streptomyces niveus NCIMB 11891 was chosen as a model. An improved DNA affinity capturing assay (DACA), combined with semi-quantitative mass spectrometry, was used to identify proteins binding to the promoter regions of the novobiocin gene cluster. Altogether, 2475 proteins were identified in DACA studies with the promoter regions of the pathway-specific regulators novE (PnovE) and novG (PnovG), of the biosynthetic genes novH-W (PnovH) and of the vegetative sigma-factor hrdB (PhrdB) as a negative control. A restrictive classification for specific binding reduced this number to 17 proteins. Twelve of them were captured by PnovH, among them, NovG, two were captured by PnovE, and three by PnovG. Unexpectedly some well-known regulatory proteins, such as the global regulators NdgR, AdpA, SlbR, and WhiA were captured in similar intensities by all four tested promoter regions. Of the 17 promoter-specific proteins, three were studied in more detail by deletion mutagenesis and by overexpression. Two of them, BxlR(Sc) and BxlR2(Sc), could be identified as positive regulators of novobiocin production in S. coelicolor M512. Deletion of a third gene, sco0460, resulted in reduced novobiocin production, while overexpression had no effect. Furthermore, binding of BxlR(Sc) to PnovH and to its own promoter region was confirmed via surface plasmon resonance spectroscopy.