Introduction: Treg migration from islet allografts to draining LN is essential for prolonging allograft survival. Lymphotoxin (LT) regulates the organization, development, and maintenance of lymphoid organs, and LT beta receptor (LTβR) signaling has been implicated in T cell migration in the thymus. However, the role of LT in T cell migration to lymph node (LN) has not been explored. Here we tested the hypothesis that LT- LTβR interactions are important for Treg migration to LN and essential for prolonging graft survival. Methods: T cell subsets were generated using Foxp3GFP mice, and purified based on CD44, CD25, and Foxp3GFP expression, or isolated directly from wild type (WT) C57BL/6 or lymphotoxin-α deficient mice (LTα KO) using CD4 and CD25 bead selection. T cells were used in transmigration assays across lymphatic endothelial SVEC4-10 or blood endothelial MS-1 cell lines. For in vivo assays, T cells were labeled with tracking dyes, injected via the foot pad, intravenously, into the ear pinna, or co-transferred with BALB/c islet allografts under the kidney capsule, and analyzed at various times and sites. LTβR fusion protein (LTβRIg) was used to block LT interactions with LTβR. Results: WT but not LTa KO Treg prolonged islet allograft function. LTα KO Treg suppressed responder T cell proliferation normally in vitro, but failed to migrate from islet grafts to dLN. WT Treg migration from footpads to popliteal dLN was inhibited by LTβRIg, while non-Treg CD4 T cell migration was unaffected. In contrast, Treg treated with LTβRIg migrated from blood directly into LN as well as controls. Both natural and induced Treg migration to lymphatics was inhibited by LTβRIg, while non Treg CD4 T cell migration was not inhibited. Treg transmigration across lymphatic endothelial SVEC4-10 cells but not blood endothelial MS-1 cells in vitro was inhibited by LTβRIg, while transmigration of non-Treg CD4 T cells was not affected. Conclusions: Treg, but not non-Treg CD4 T cells, rely upon stimulation of LTβR on lymphatic endothelium for migration to LN via afferent lymphatics. This interaction is not required for LN entry from blood through HEV. This reveals a previously undescribed role for LTβR in regulating Treg entry into LN. Since Treg are specialized to engage afferent lymphatics to enter LN and be fully suppressive, modulating this pathway may allow targeted disruption or enhancement of Treg migration to LN and suppress or enhance immunity or tolerance.