Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)(2) fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.