We measured the effect of a model membrane-binding protein on line tension and morphology of phase-separated lipid-bilayer vesicles. We studied giant unilamellar vesicles composed of a cholesterol/dioleoylphosphatidylcholine/palmitoylsphingomyelin mixture and a controlled mole fraction of a Ni-chelating lipid. These vesicles exhibited two coexisting fluid-phase domains at room temperature. Owing to the line tension, a, between the two phases, the boundary between them was pulled like a purse string so that the smaller domain formed a bud. While observing the vesicles in a microscope, histidine-tagged green fluorescent protein was added, which bound to the Ni-chelating lipid. As protein bound, the vesicle shape changed and the length of the phase boundary increased. The change in morphology was attributed to a reduction of a between the two phases because of preferential accumulation of histidine-tagged green fluorescent protein Ni-chelating lipid clusters at the domain boundary. Greater reductions of a were found in samples with higher concentrations of Ni-chelating lipid; this trend provided an estimate of the binding energy at the boundary, approximately k(B)T. The results show how domain boundaries can lead to an accumulation of membrane-binding proteins at their boundaries and, in turn, how proteins can alter line tension and vesicle morphology.