The Sleeping Beauty (SB) transposase reconstructed from salmonid fish has high transposition activity in mammals and has been a useful tool for insertional mutagenesis and gene delivery. However, the transposition efficiency has varied significantly among studies. Our previous study demonstrated that the introduction of methylation into the SB transposon enhanced transposition, suggesting that transposition efficiency is influenced by the epigenetic status of the transposon region. Here, we examined the influence of the chromatin status on SB transposition in mouse embryonic stem cells. Heterochromatin conformation was introduced into the SB transposon by using a tetracycline-controlled transrepressor (tTR) protein, consisting of a tetracycline repressor (TetR) fused to the Kruppel-associated box (KRAB) domain of human KOX1 through tetracycline operator (tetO) sequences. The excision frequency of the SB transposon, which is the first step of the transposition event, was enhanced by approximately 100-fold. SB transposase was found to be colocalized with intense DAPI (4′,6′-diamidino-2-phenylindole) staining and with the HP1 family by biochemical fractionation analyses. Furthermore, chromatin immunoprecipitation analysis revealed that SB transposase was recruited to tTR-induced heterochromatic regions. These data suggest that the high affinity of SB transposase for heterochromatin conformation leads to enhancement of SB transposition efficiency.