The role of the host plasminogen activation system in transmission of and invasion by Borrelia burgdorferi, the tick-borne spirochetal agent of Lyme disease, was investigated using plasminogen (Plg)-knockout mice. PLG was not detected in spirochetes from unfed ticks, but binding occurred as ticks fed on the host's blood. Plasminogen activators were derived from the host blood meal. PLG was required for efficient dissemination of B. burgdorferi within the tick and for enhancement of spirochetemia in mice but was not critical for transmission and infection. These results provide evidence for a bacterium using a vertebrate protease to disseminate in an invertebrate vector and underscores the interplay among vector, pathogen, and host in promoting the life cycle and disease.