Lyme disease is the most common tick-borne illness in the United States. In this paper we explore the contribution of Ixodes scapularis ticks to the pathogenicity of Borrelia burgdorferi in mice. Previously we demonstrated that an isolate of B. burgdorferi sensu stricto (designated N40), passaged 75 times in vitro (N40-75), was infectious but was no longer able to cause arthritis and carditis in C3H mice. We now show that N40-75 spirochetes can readily colonize I. scapularis and multiply during tick engorgement. Remarkably, tick-transmitted N40-75 spirochetes cause disease in mice. N40-75 spirochetes isolated from these animals also retained their pathogenicity when subsequently administered to mice via syringe inoculation. Array analysis revealed that several genes associated with virulence, including bba25, bba65, bba66, bbj09, and bbk32, had higher expression levels in the tick-passaged N40-75 spirochete. These data suggest that transmission of a high-passage attenuated isolate of B. burgdorferi by the arthropod vector results in the generation of spirochetes that have enhanced pathogenesis in mice.