<p>Membrane fusion is fundamental for many biological processes, including subcellular compartmentalization, cell growth, and exo- and endocytosis. It also plays a key role in the fabrication of artificial membrane structures, particularly vesicles. However, replicating membrane fusion in vitro under neutral pH conditions without prior modification of lipid vesicles with fusion-promoting biomolecules has been a significant challenge, hampering the development of biomimetic fusion systems and their potential applications. Here, we reveal diphtheria toxin T-domain as a fusogenic tool that renders membrane modifications unnecessary. The T-domain spontaneously interacts with anionic nanosized unilamellar vesicles at neutral pH in a non-disruptive manner. We demonstrate that membrane-associated T-domain mediates the adsorption of these small unilamellar vesicles to a glass substrate, causing the vesicle membranes to fuse into large and giant unilamellar vesicles. We show that basic amino-acid residues play a critical role in this membrane fusion. Our findings hold significant implications for the development of lab-on-a-chip devices, biosensors, active surfaces and similar technologies.</p><p></p>