Lyme borreliosis is one of the major tick-borne diseases in Europe. Events of the translocation of Borrelia across the blood-brain barrier (BBB) involve multiple interactions between borrelial surface proteins and receptors on the human brain microvascular endothelial cells (hBMECs). The first aim of this study was set to identify borrelial proteins plausibly interact with hBMECs. To achieve this, the surface proteome of live B. garinii was crosslinked with biotin before its incubation with hBMECs. The interacting proteins were recovered by affinity purification, followed by SWATH-MS, where 24 interacting proteins were identified. These candidates were grouped into outer membrane proteins (n = 12) and inner membrane proteins (n = 12) based on subcellular localization. Six proteins namely, LysM domain protein, BESBP-5, Antigen S1, CRASP-1 (Bg071), Erp23 protein and Mlp family Lipoprotein were selected to produce their recombinant forms and all proteins experimentally showed interaction with hBMECs by ELISA and immunocytochemistry. The second aim was set to identify potential receptors on hBMECs interacting with borrelial surface proteins. To achieve this, biotinylated surface proteins of hBMECs were used for incubation with live B. garinii. The interacting proteins were captured by affinity purification, followed by LC-MS. Eleven potential host receptors were detected. It is known that ligand-receptor interactions trigger numerous signaling cascades in the hBMECs and pathogen can exploit them to traverse across the BBB. Thus, the third aim was focused to map the complete picture of signaling events in hBMECs induced by B. garinii. To achieve this aim, RNA sequencing was used, where 11 398 genes were mapped and among them 295 genes were differentially expressed genes (DEGs, 251 upregulated genes and 44 downregulated genes) in hBMECs challenged by live B. garinii. The results obtained from RNA-seq were validated by qRT-PCR. The gene ontology analysis revealed the participation of the DEGs in a plethora of the biological processes, but in this study biological processes that involved in borrelial translocation across the BBB were focused. Expression of several non-protein coding genes were also evoked. The data obtained in this study were revealed the borrelial interacting surface proteins and potential hBMECs receptors that participate in host-pathogen interactions and show us the signaling cascades, that can be important in mechanisms of B. garinii to cross the BBB.