RNA interference (RNAi) is an evolutionary conserved cellular process for the regulation of gene expression. In mammalian cells, RNAi is induced via short (21–23nt) duplexes of RNA, termed small interfering RNA (siRNA), that can elicit highly sequence-specific gene silencing. However, synthetic siRNA duplexes are polyanionic macromolecules that do not readily enter cells and typically require the use of a delivery vector for effective gene silencing in vitro and in vivo. Choice of delivery system is usually made on its ability to enhance cellular uptake of siRNA. However, recent gene expression profiling (toxicogenomics) studies have shown that separate from their effects on cellular uptake, delivery systems can also elicit wide ranging gene changes in target cells that may impact on the ‘off-target’ effects of siRNA. Furthermore, if delivery systems also alter the expression of genes targeted for silencing, then siRNA activity may be compromised or enhanced depending on whether the target gene is up-regulated or down-regulated respectively. Citing recent examples from the literature, this article therefore reviews the toxicogenomics of non-viral delivery systems and highlights the importance of understanding the genomic signature of siRNA delivery reagents in terms of their impact on gene silencing activity and specificity. Such information will be essential in the selection of optimally acting siRNA-delivery system combinations for the many applications of RNA interference.