The Bloom helicase(BLM) gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes(G4), which are noncanonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer(smFRET) to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action: when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately. The Bloom helicase (BLM) gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes (G4), which are noncanonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer (smFRET) to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action: when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.