Here we report a simple and efficient method for nonviral gene transfer using liposomes which have envelope protein of vesicular stomatitis virus (VSV) on their surface (VSV-liposomes). We prepared VSV-liposome by fusing simple liposomes with VSV particles. The density of VSV-liposome fusion products was intermediated between that of liposomes and that of VSV particles. Furthermore, VSV-liposome fusion products included both viral proteins and lipids from liposomes, and were confirmed to be fusion products, but not adsorptive products, by the resonance energy transfer fusion assay. To evaluate whether these particles can efficiently introduce their internal contents into the cytoplasm of mammalian cells, we examined the delivery of fragment A of diphtheria toxin (DTA) by VSV-liposomes into the cytoplasm of FL cells. We found that VSV-liposomes encapsulating DTA were highly cytotoxic to the cells, while empty VSV-liposomes and plain liposomes encapsulating DTA were not, suggesting that VSV-liposomes delivered DTA into cytoplasm. Consistent with this, the cells cultured with plasmid DNA entrapped in VSV-liposomes and coding for firefly luciferase showed significant luciferase expression, whereas cells culture with plasmid DNA in plain liposomes and plasmid DNA-cationic liposomes complex did not. Thus, VSV-liposomes function as a simple and efficient nonviral vector for the delivery of DNA.