During the natural enzootic life cycle of (also known as ), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. of encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the enzootic cycle. To study the role of BB0345 within mammals, we generated a mutant and assessed its virulence potential in immunocompetent mice. The mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. growth curve analyses revealed that the mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.