The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G that, when subjected to a pH 6.0, induces membrane fusion between virus envelope and endosome membrane. In a previous study, we showed that the sequence corresponding amino acid 145–164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. In this work, we present the AFM imaging mode as an effective approach to study the interaction of this fusion peptide on lipid membrane. In the last few years, the Atomic Force Microscopy (AFM) has been used to study biological samples under physiological conditions through force measurements and image acquisition at high resolution. However, this is the first time that vesicles fusion is observed through this technique. Phosphatidylcholine:phosphatidylserine (PC:PS) membranes were prepared by fusion of small unilamellar vesicles by calcium addition. Lipid membrane and small vesicles were imaged over freshly cleaved mica. All the samples were imaged in buffer solution without calcium, in the absence and in the presence of the peptide [145-164]. The peptide [145-164] solution was shown to induce rapid perturbation on membrane, and shifting the size and shape of vesicles at pH 6.0. Interactions of the peptide molecules with the head of the phospholipids could create membrane destabilization, elucidating what we have observed in both cases. No fusion or difference at the bilayer was observed at pHs 7.5 or pH 4.0. These results were directly correlated with fusogenic activity of the peptide as determined using fluorescently labeled PC:PS liposomes.