Significance: G-quadruplexes (G4) are 4-stranded nucleic acid secondary structures. MYC is a critical oncogene with a G4 in its promoter (MycG4), which acts as a transcription silencer. MycG4 is very stable and the pathological activation of MYC in cancers requires its active unfolding. We reveal herein that DDX5, a founding member of the DEAD-box RNA helicase family, can unfold DNA G4s. The unfolding mechanism of DDX5 is distinct from previously characterized G4 helicases. Importantly, DDX5 activates MYC gene transcription by proficiently unfolding the promoter MycG4. DDX5 is overexpressed in cancers and the DDX5–MycG4 interaction can be inhibited by small molecules to downregulate MYC . Therefore, our results suggest a new molecular target to suppress MYC for cancer intervention.