We report the coupling of phase separation on a phospholipid membrane with a structural transition of giant DNA. We performed single-DNA measurements in the presence of cell-sized giant unilamellar vesicles (GUV) composed of dioleoylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), and cholesterol. We observed the effect of phase separation of the membrane by changing PE from dioleoylphosphatidylethanolamine (DOPE) to dipalmitoylethanolamine (DPPE), which corresponds to a change from a homogeneous single phase to two segregated phases of liquid-ordered and liquid-disordered states on the membrane. In the homogeneous membrane, DNA molecules exhibited a compact conformation in aqueous solution containing a physiological concentration of Mg2+ and polyamine, without attaching to the membrane surface. In contrast, in the membrane that was segregated into two domains, DNA molecules were adsorbed onto the liquid-ordered phase that was rich in DPPE by taking an elongated conformation.Keywords (keywords): giant unilamellar vesicle; liposome; membrane domain; phosphatidylethanolamine; DNA−phospholipid interaction; conformational transition of DNA