Summary The delivery of DNA to mammalian cells is of critical importance to the development of genetic vaccines, gene replacement therapies and gene silencing. For these applications, targeting, effective DNA transfer and vector safety are the major roadblocks in furthering development. In this report, we present a novel DNA delivery vehicle that makes use of protoplasted, achromosomal bacterial minicells. Transfer of plasmid DNA as measured by green fluorescent protein expression was found to occur in as high as 25% of cultured Cos‐7 cells when a novel chimeric protein containing the D2–D5 region of invasin was expressed and displayed on the surface of protoplasted minicells. Based on endoplasmic reticulum stress and other responses, protoplasted minicells were non‐toxic to recipient eukaryotic cells as a consequence of the transfection process. Taken together, these results suggest that bacterial minicells may represent a novel and promising gene delivery vehicle.