The Sleeping Beauty (SB) transposable element shows efficient transposition in human cells, and provides long-term transgene expression in preclinical animal models. Random chromosomal insertion of SB vectors represents a safety issue in human gene therapeutic applications, due to potential genotoxic effects associated with transposon integration. We investigated the transcriptional activities of SB in order to assess its potential to alter host gene expression upon integration. The untranslated regions (UTRs) of the transposon direct convergent, inward-directed transcription. Transcription from the 5'-UTR of SB is upregulated by the host-encoded factor high-mobility group 2-like 1 (HMG2L1), and requires a 65-base pair (bp) region not present in commonly used SB vectors. The SB transposase antagonizes the effect of HMG2L1, suggesting that natural transposase expression is under a negative feedback regulation. SB transposon vectors lacking the 65-bp region associated with HMG2L1-dependent upregulation exhibit benign transcriptional activities, at a level up to 100-times lower than that of the murine leukemia virus (MLV) long terminal repeat (LTR). Incorporation of chicken beta-globin HS4 insulator sequences in SB-based vectors reduces the transactivation of model promoters by transposon-borne enhancers, and thus may lower the risk of transcriptional activation of host genes situated close to a transposon insertion site.