Abstract: In this study, the features of SB-mediated integration were examined in grass carp (Ctenopharyngodon idellus) kidney cells (CIK). Two expression systems were constructed: the transposase and transposon reporters were separated into two vectors designated as SB trans-configuration (trans) system, whereas the transposase and transposon cassettes were combined in one vector named as SB cis-configuration (cis) system. The vectors were transfected into CIK cells, and then the transfection and integration efficiency of DsRed were investigated by fluorescence microscope, flow cytometry, and quantitative RT-PCR after 2 days or 4 weeks of puromycin selection. In addition, the unknown sequences within the integration regions were also characterized by hiTAIL-PCR. The results showed that the integration efficiency of SB trans system was much higher than the cis system; the highest integration efficiency of DsRed was observed when the ratio of transposon versus transposase was 1﹕2; the transposon was inserted randomly into TA dinucleotides with preference. In conclusion, this study demonstrated that optimizing the ratio of SB transposon versus transposase increased the exogenous gene integration efficiency and generated mutant cells rapidly.