<p>Although the contours of the dissemination pathways of human pathogenic spirochetes in the vertebrate hosts are known, detailed high-resolution information on these processes remain lacking. In this study, we establish an efficient serial block-face scanning electron microscopy workflow incorporating semi-automatic AI-driven segmentation to investigate the architecture of early events following the deposition of <i>Borrelia burgdorferi</i> at the tick bite site in mice. We capture evidence of <i>Borrelia</i> penetrating the lymphatic endothelium via both transcellular and paracellular routes and observe its early presence within the lumen of the lymphatic vessel. The multistep process of transcellular migration is documented in detail, showing sequential invagination and encasement of shorter <i>Borrelia</i> segments by the lymphatic endothelial cells during intravasation. Our findings reveal that the first contact of <i>B. burgdorferi</i> and blood vessels is not random but involves close interactions with pericytes. We also capture the infiltration of immune cells in the skin and their interactions with invading bacteria. Altogether, these observations suggest that <i>Borrelia</i> strategically targets vascular regions with lower mechanical resistance to breach the endothelial barrier, thereby enhancing its dissemination.</p>