Borrelia burgdorferi, the bacterial agent of Lyme disease, induces the production of type I IFNs by human DCs through TLR7 and TLR9 signaling. This type I IFN response occurs in a genotype-dependent manner, with significantly higher levels of IFN-α elicited by B. burgdorferi strains that have a greater capacity for causing disseminated infection. A B. burgdorferi strain that was previously shown to induce IFN-α was found to elicit significantly higher levels of IDO1 protein and its downstream metabolite, kynurenine, compared with a B. burgdorferi mutant that lacks a single linear plasmid (lp36); this mutant is unable to induce IFN-α and is severely attenuated for infectivity in mice. Production of IDO by mDC and pDC populations, present within human PBMCs, was concomitant with increased expression of the DC maturation markers, CD83 and CCR7. The defects in IDO production and expression of CD83 and CCR7 could be restored by complementation of the mutant with lp36. Maximal IDO production in response to the wild-type strain was dependent on contributions by both type I IFN and IFN-γ, the type II IFN. Induction of IDO was mediated by the same TLR7-dependent recognition of B. burgdorferi RNA that contributes to the production of type I IFNs by human DCs. The ability of IFN-α-inducing B. burgdorferi strains to stimulate production of IDO and kynurenines may be a mechanism that is used by the pathogen to promote localized immunosuppression and facilitate hematogenous dissemination.