The pathogen of Lyme disease, Borrelia burgdorferi, persists in a natural tick-rodent infection cycle. An assessment of microbial transcriptome in vivo encoding selected membrane proteins shows that bba52 is upregulated during pathogen transmission. Deletion of bba52 in infectious B. burgdorferi did not alter the outcome of murine inflammation or long-term pathogen persistence in mice or ticks. However, the bba52 mutant was impaired for transmission between feeding ticks and mice, phenotypic defects that could be rescued when bba52 was genetically restored to the original genomic locus. I show that BBA52 is a surface-exposed outer membrane protein. As BBA52 is highly immunogenic, I next assessed whether BBA52 could serve as a potential candidate for transmission-blocking vaccines against spirochete infection. Passive transfer of BBA52 antibodies into the ticks did not interfere with microbial persistence in unfed ticks but blocked pathogen transmission from feeding ticks to murine hosts. More importantly, active immunization of mice with recombinant BBA52 protein significantly blocked B. burgdorferi transmission from ticks to naïve murine hosts. As BBA52 antibodies lacked detectable borreliacidal activities, their interference with spirochete survival in vivo could result from the inhibition of BBA52 function, such as vector-pathogen interaction. By far-western analysis using tick gut proteins, I found that BBA52 interacts with a ~ 35 kDa tick gut protein. I also show that BBA52 forms distinct homo-oligomer in borrelial cells and also interacts with two proteins of B. burgdorferi. These BBA52 interacting proteins possess approximate molecular weight of 33 kDa and 25 kDa and are located in the outer membrane and protoplasm, respectively. Taken together, these studies suggested the remarkable existence of B. burgdorferi surface antigens that are differentially expressed in vivo and support microbial transitions between hosts and the vector. Identification and characterization of novel vector-specific and feeding induced borrelial antigens could contribute to the development of transmission-blocking vaccines against Lyme disease.