Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine, and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors, and potential risks associated with random genomic insertion.