Borrelia burgdorferi, the etiologic agent of Lyme disease, persists in both an arthropod vector and vertebrate hosts, usually wild rodents. Analysis of the B. burgdorferi transcriptome in vivo indicates that the bb0365 gene is markedly induced as spirochetes enter the feeding ticks from infected mice. To understand the importance of the bb0365 gene product in the spirochete life cycle, we inactivated this gene in an infectious isolate of B. burgdorferi B31. BB0365-deficient spirochetes were fully pathogenic in mice and survived in diverse murine tissues. When naive ticks engorged on spirochete-infected mice, the B. burgdorferi bb0365 mutant entered ticks but had a markedly decreased survival rate compared with wild type B. burgdorferi. BB0365 therefore is not necessary for B. burgdorferi persistence in the vertebrate host but is required for survival of the Lyme disease agent within the feeding arthropod vector, and strategies for interfering with this gene may potentially interrupt the B. burgdorferi life cycle.