The Sleeping Beauty (SB) transposon (Tn) is a recently developed non-viral vector that can mediate insertion of therapeutic transgenes into mammalian genomic DNA. In this system, the relevant transgene is placed between the SB-Tn terminal flanking inverted repeats/direct repeats (IR/DRs), which are essential binding sites for the obligate non-native catalytically active SB transposase. Transposition, i.e. molecular translocation of the SB-Tn from the plasmid vector into the host's genome requires expression of the SB transposase in the host cell, to mediate the excision and insertion of the IR/DR flanked transgene. However, foreign DNA elements introduced into the mammalian genome tend to invoke a host-defensive mechanism resulting in epigenetic changes, such as DNA methylation. Methylation of DNA is associated with the transcriptional inactivation of many mammalian genes, and methylation dependent silencing of the introduced transgene has been a key factor in the failure of gene replacement therapy. The aim of this study was to investigate the DNA methylation status of SB-Tns as well as the flanking genomic regions following transposition into the mouse genome. SB-Tns carrying a human keratin 14 (K14) promoter expressing a mouse agouti transgene was used to create transgenic mouse lines via transposition of the SB-Tn transposons. DNA isolated from tail clips of transgenic or wt control mice were subjected to bisulfite-mediated genomic sequencing. DNA methylation profiles were determined for the human K14 promoter, mouse agouti transgene, and the genomic regions flanking the Tn insertion sites. The results indicated that only a very low level of DNA methylation was present at the human K14 promoter CpG islands in the transposed SB-Tns, irrespective of whether expression of the agouti transgene was observed. The agouti transgene was almost devoid of methylated CpGs regardless of the loci of SB insertion, or mode of insertion by either random integration or SB-mediated transposition. We also analyzed the DNA methylation pattern at two different chromosomal loci flanking independent SB-mediated insertions of the Tns. Both chromosomal loci flanking the SB-Tn insertion sites showed identical DNA methylation patterns as those observed in the wt mice. This indicates that the SB-mediated transposition does not induce novel epigenetic DNA methylation of the flanking genomic region. In addition, expression of the agouti transgene ranging from mice of black to yellow phenotype seems to be regulated by mechanisms other than DNA methylation. In conclusion, the translocation of the SB-Tn does not invoke a significant level of DNA methylation of the cargo DNA. In fact, the level of transgene expression appears to be independent of promoter or coding sequence CpG methylation status in the agouti transgenic mice. Moreover, in contrast to integrating viral vectors, insertion of a foreign SB-Tn element did not alter the DNA methylation status in the flanking genomic regions. Thus, the SB-Tn system provides an alternative method of stable gene transfer that circumvents a significant safety concern associated with gene replacement mediated by viral vectors.