Somatic cell gene transfer has permitted inducible gene expression in vivo through coinfection of multiple viruses. We hypothesized that the highly efficient plasmid-based piggyBac transposon system would enable long-term inducible gene expression in mice in vivo. We used a multiple-transposon delivery strategy to create a tetracycline-inducible expression system in vitro in human cells by delivering the two genes on separate transposons for inducible reporter gene expression along with a separate selectable transposon marker. Evaluation of stable cell lines revealed 100% of selected clones exhibited inducible expression via stable expression from three separate transposons simultaneously. We next tested and found that piggyBac-mediated gene transfer to liver or lung could achieve stable reporter gene expression in mice in vivo in either immunocompetent or immune deficient animals. A single injection of piggyBac transposons could achieve long-term inducible gene expression in the livers of mice in vivo, confirming our multiple-transposon strategy used in cultured cells. The plasmid-based piggyBac transposon system enables constitutive or inducible gene expression in vivo for potential therapeutic and biological applications without using viral vectors.