[en] Highlights: • Many G-tracts with G-quadruplex potential exist in negative strand of BAP1 promoter. • Multiple approaches were used to confirm G-quadruplex formation in BAP1 promoter. • Strikingly, G-quadruplex disruption reduced BAP1 promoter-mediated gene expression. • DNA helicases CHD2 and CHD7 potential unwind G-quadruplexes in BAP1 promoter. Accumulating evidence suggests a key role of BAP1 in oncogenesis, but mechanisms regulating BAP1 gene expression remain unexplored. In this report, we revealed that the BAP1 promoter contains multiple G-tracts in its negative strand with high potential of forming G-quadruplex (G4) structures. In circular dichroism studies, synthesized oligonucleotides within these G-rich regions upstream the BAP1 transcription start site showed molar ellipticity at specific wavelengths characteristic of G4 structures. Analyses of these oligonucleotides by native polyacrylamide gel electrophoresis revealed formation of multiple types of G4 structures. In reporter assays, mutations or deletion of predicted G4 structures reduced BAP1 promoter activity. Additionally, DNA helicases CHD2 and CHD7 could reduce BAP1 promoter activity, likely through unwinding its G4 structures.