Introduction: Homeostatic circulation of immune cells via blood and lymph is integral to adaptive immunity. Afferent lymphatics in peripheral tissues constitutively produce a CCL21 gradient to guide dendritic cells and T cells to lymphatic vessels and then to draining lymph nodes (dLN). VEGF-C/VEGFR-3 are, respectively, the major growth factor and its receptor for lymphatic endothelial proliferation. We hypothesized that these molecules may also regulate chemokine gradients and lymphatic migration. Methods: To assess migration from tissues to dLN, CFSE-labeled CD4+ T cells were injected into the foot pad and migration to the draining popliteal LN quantified. To assess migration into afferent lymphatics, T cells were injected into the ear pinna and visually quantified by whole mount immunohistochemistry (IHC). Anti-VEGFR-3 or control mAb were injected with the T cells. Results: The number of transferred CD4+ T cells detected in dLN peaked at 12 hours after tissue transfer, and was significantly inhibited by over 95% with anti-VEGFR-3. Whole mount tissue IHC showed that anti-VEGFR-3 prevented migration of CD4+ T cells into the lymphatic lumen and caused tissue retention, with a greater than 3-fold increase in the number of cells remaining in the tissues. IHC also demonstrated that anti-VEGFR-3 abolished the CCL21 gradient around the lymphatic vessels, yet flow cytometry and qRT-PCR showed that CCL21 production were not inhibited. Heparan sulfate (HS), a major component of the proteoglycan surrounding lymphatics, is known to be critical to establish the CCL21 gradient by immobilizing chemokines. IHC showed that anti-VEGFR-3 down-regulated HS expression around the lymphatic vessels. Heparanase (HPSE) is known to regulate HS expression, and neutralization with anti-HPSE Ab prevented anti-VEGFR-3 induced reduction of HS and dispersion of the CCL21 gradient. VEGFR-3 signaling is dependent on PI3Ka, and a PI3Ka inhibitor likewise inhibited CCL21 and HS expression, while a PI3K activator prevented the effects of anti-VEGFR-3. Conclusion: VEGF-C/VEGFR-3 signaling through PI3Ka regulates the activity of HPSE, which in turn regulates the HS and CCL21 gradients around tissue afferent lymphatics. These molecules and their functional and physical arrangement regulate CD4+ T lymphocyte migration in tissues to dLN. These represent a new target to influence immunity and tolerance.