Lyme disease, caused by the spirochete Borrelia burgdorferi and closely related Lyme Borrelia, is the most prevalent tick-borne illness in the northern hemisphere. An important pathway for B. burgdorferi dissemination is its interaction with, and traversal of the vascular endothelium, a process that is not well understood and is mediated by spirochete surface adhesins. We show here that infection-induced activation of the endothelium in BALB/c mice results in new B. burgdorferi-endothelial interactions, indicating the presence of spirochete factors that interact specifically with activated endothelial cells. We show that these interactions are mediated by spirochetal surface proteins whose synthesis is dependent upon B. burgdorferi host adaptation. We used intravital microscopy and a functional gain approach to assess the binding of spirochetes that withstand the shear force of blood flow in post-capillary venules of living mice. We identified five previously undescribed, shear force-resistant adhesins that selectively mediate binding to activated endothelium (BBA66, P66, BBA36, BBA07 and DbpA) and interact with activation-induced endothelial surface changes. Two of these adhesins (P66 and DbpA) have been implicated in the spirochete extravasation process. We also identified seven previously undescribed shear force-resistant adhesins that target pre-activated endothelium (BBA04, BBK53, BBK07, BBA65, BB0844, ErpK and OspC). Three of these (BBK53, ErpK and OspC), display reduced binding to activated endothelium, a property that may facilitate the multi-step pathway of vascular transmigration. In particular, OspC has been previously implicated in spirochete extravasation. In summary, our results reveal a dynamic interaction network between the spirochete and the endotheliu