The binding characteristics and molecular mechanism of the interaction between a typical ionic liquid (IL), 1-butyl-3-methylimidazolium chloride ([bmim]Cl), as a green solvent and DNA were investigated for the first time by conductivity measurements, fluorescence spectroscopy, dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), circular dichroism spectroscopy, 3 T nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy, isothermal titration calorimetry (ITC), and quantum chemical calculations. It was found that the critical aggregation concentration of [bmim]Cl is decreased in the presence of DNA, and the addition of [bmim]Cl induced a continuous fluorescence quenching of the intercalated probe ethidium bromide (EtBr), indicating that the interaction between the ionic liquid and DNA is sufficiently strong to exclude EtBr from DNA. DLS results show that [bmim]Cl can induce a coil-to-globule transition of DNA at a low IL concentration, which was confirmed by the cryo-TEM images of DNA-IL complexes. With [bmim]Cl added, the resulting globular DNA structures and the extended DNA coils are first compacted, and then grow in size. During the binding process, DNA maintains the B-form, but the base packing and helical structure of DNA are altered to a certain extent. The P-31 NMR and IR spectra indicate that the cationic headgroups of bmim(+) groups interact with the phosphate groups of DNA through electrostatic attraction, and the hydrocarbon chains of bmim(+) groups interact with the bases through strong hydrophobic association. ITC results reveal the interaction enthalpy between [bmim]Cl and DNA and show that the hydrophobic interaction between the hydrocarbon chains of [bmim]Cl and the bases of DNA provides the dominant driving force in the binding. On the basis of quantum chemical calculations, it can be inferred that at a low IL concentration, the cationic headgroups of [bmim]Cl would be localized within several angstroms of the DNA phosphates, whereas the hydrophobic chains would be arranged parallel to the DNA surface. When the IL concentration is above 0.06 mol/L, the cationic headgroups are near DNA phosphates, and the hydrocarbon chains are perpendicularly attached to the DNA surface.