Mammalian cells express transfected plasmid DNA transiently, but the underlying mechanisms are not well known. Plasmid DNA was found to be captured in the cytoplasmic, (ER)Endoplasmic reticulum-enwrapped exclusome, which clusters both endogenous and exogenous extrachromosomal DNA. Exclusome-envelope formation corresponded to early but not late steps of nuclear-envelope formation. This work suggests that a cell-autonomous genome protection system recognizes, collects, and maintains extrachromosomal DNA in somatic mammalian cells.