Innate immune responses are triggered when pattern-recognition receptors recognize specific conserved patterns on pathogens. The most extensively studied pattern-recognition receptors are toll-like receptors (TLRs), which are comprised of 11 different receptors, named TLR1-11. TLRs recognize motifs that are found on a wide range of pathogens, and activation of TLRs results in the production of large amounts of type I interferons and several proinflammatory cytokines. These cytokine responses are important in controlling pathogen replication and they also provide an initiation signal for the adaptive immune response. Although numerous manuscripts have reviewed the important role of TLRs in host defense against wild-type viruses, bacteria, and/or their subcomponents, none have focused on how TLRs recognize commonly utilized gene delivery vehicles such as adenovirus and adeno-associated virus (AAV) vectors. In this review, we discuss our understanding of how TLRs are activated by gene transfer vectors.