The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. ModifiedpiggyBac(PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies ofPBamong different cell lines have restricted its application. We discovered that the DNA-PK complex facilitatesPBtransposition by binding toPBtransposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK componentsKu70,Ku80, andDNA-PKcsOverexpression or knockdown of DNA-PK components enhances or suppressesPBtransposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency ofPBis significantly reduced inKu80heterozygous mutant mice, confirming the role of DNA-PK in facilitatingPBtransposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of thePBtransposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitatingPBtransposition and suggest a transposition-promoting manipulation by enhancing the interaction of thePBends. Consistent with this, deletions shortening the distance between the twoPBends, such asPBvectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.