Rats are an important laboratory animal for biomedical research. Though rats have some physiology and genetic similarities to human, several technical issues such as delicate in vitro culture system and low survival rate after pronuclear microinjection have hindered the development of transgenic rat generation. Accordingly, in this study, to produce transgenic rat, we established transposon-mediated insertional mutagenesis by cytoplasmic microinjection. The sleeping beauty transposon (SB) and SB-transposase recognize the precise genome integration into a TA nucleotide by ‘cut-and-paste’ mechanism. It mediates stable integration and reliable long-term expression. DNA, 0.4ng/ul SB vector (IR/DR-EF1a-eGFP-2A-IL2-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo and one transgenic rat was generated after full-term gestation. To confirm the genome insertion, GFP was detected by PCR. Further, this method was applied to generate transgenic rats producing Cas9 protein. DNA, 0.4ng/ul SB vector (IR/DR-CAG-Cas9-2A-eGFP-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo. Some of the in vitro cultured embryos showed GFP positive at blastocyst stage and Cas9 sequence was detected by PCR. One stillbirth pup was born to date and genome PCR on Cas9 was positive. In summary, the SB transposon system could be a highly effective method that contribute to the production of transgenic rats. If the protocols will be optimized, we successfully generated efficiently transgenic rats for human models by SB system. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972) and the Technology Development Program (S2566872) by MSS.