Borrelia burgdorferi is the spirochetal agent of Lyme disease, a multisystemic disorder characterized by inflammation. Using global transcriptional profiling, we characterized the response of human PBMCs exposed to B. burgdorferi in an ex vivo coculture system. The expression profiles induced by B. burgdorferi were marked by the intense up-regulation of IFN-responsive transcripts and transcripts involved in the JAK/STAT signaling pathway. Transcript levels of IFN-alpha, IFN-beta, and IRF7, and protein concentrations of IFN-alpha, were significantly elevated relative to those in unstimulated PBMCs. The induction of IFN-alpha was completely dependent upon phagocytosis of B. burgdorferi. Addition of a soluble type I IFN receptor, B18R, did not abolish the induction of IFN-inducible genes, indicating that B. burgdorferi directly elicits enhanced expression of these genes independently of type I IFN feedback signaling. Inhibitors of either TLR7 or TLR9 significantly reduced B. burgdorferi-stimulated IFN-alpha protein expression and transcription of IFN-induced genes. Simultaneous inhibition of both TLR7 and TLR9 completely abrogated IFN-alpha induction. The IFN-alpha-producing populations in PBMCs were identified as plasmacytoid dendritic and CD14(+)CD11c(+) cells. These results reveal a TLR7/9-dependent signaling pathway used by human PBMCs to initiate a type I IFN response to the extracellular bacterium B. burgdorferi.