Borrelia burgdorferi (Bb) is the spirochetal agent of Lyme disease, the most common tick-transmitted infection in the United States. This extracellular pathogen initiates a type I interferon (IFN) response following phagocytosis and recognition of live Bb by toll-like receptors (TLRs) 7 and 9. To identify the ligands of Bb that induce the type I IFN response in human cells, cellular components of Bb were delivered into peripheral blood mononuclear cells (PBMCs) by encapsulation with DOTAP. Type I IFN-responsive genes (MX1, OAS1, IRF7) were significantly induced by RNA, DNA, and cell lysates relative to unstimulated PBMCs (p<0.05 for all). Unencapsulated lysate or lysate treated with both RNase and DNase were unable to elicit this response. In contrast, proteinase K treatment of lysate had no effect. Purified, DOTAP-encapsulated Bb DNA and RNA induced production of significant levels of IFN-α (p<0.01). The IFN response to RNA was abolished with RNase treatment or by specific inhibition of TLR7 (p<0.01). This demonstrates that Bb RNA is the ligand for TLR7 in human cells, contributing to IFN-α production and induction of IFN-responsive genes. These results support prior data demonstrating that the type I IFN response requires phagocytosis and is not mediated by a cell surface receptor in human cells. Taken together, these data demonstrate that RNA (via TLR7) and DNA, but not protein, are the Bb cellular components that initiate the type I IFN response in human PBMCs.