Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.