Transposable elements have been routinely used for genetic manipulation in lower organisms, including generating transgenic animals and insertional mutagenesis. In contrast, the usage of transposons in mice and other vertebrate systems is still limited due to the lack of an efficient transposition system. We have tested the ability of piggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, to transpose in mammalian systems. We show that PB elements carrying multiple genes can efficiently transpose in human and mouse cell lines and also in mice. PB permits the expression of the marker genes it carried. During germline transposition, PB could excise precisely from original insertion sites and transpose into the mouse genome at diverse locations, preferably transcription units. These data provide a first and critical step toward a highly efficient transposon system for a variety of genetic manipulations including transgenesis and insertional mutagenesis in mice and other vertebrates.