Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumoral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilising compound, berberine, to these non-canonical genetic structures using UV-Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.