The beneficial effects of checkpoint blockade in tumor immunotherapy are limited to patients with increased tumor-infiltrating lymphocytes (TILs). Delineation of the regulatory networks that orchestrate the presence of TILs holds great promise for the design of effective immunotherapies. Podoplanin/gp38 (PDPN)-expressing lymph node stromal cells (LNSCs) are present in tumor stroma; however, their effect in the regulation of TILs remains elusive. Herein we demonstrate that intratumor injection of ex-vivo-isolated PDPN⁺ LNSCs into melanoma-bearing mice induces elimination of TILs and promotes tumor growth. In support, PDPN⁺ LNSCs exert their function through direct inhibition of CD4⁺ T cell proliferation in a cell-to-cell contact independent fashion. Mechanistically, we demonstrate that PDPN⁺ LNSCs mediate T cell growth arrest and induction of apoptosis to activated CD69⁺CD4⁺ T cells. Importantly, LTbR-Ig-mediated blockade of PDPN⁺ LNSCs expansion and function significantly attenuates melanoma tumor growth and enhances the infiltration and proliferation of CD4⁺ TILs. Overall, our findings decipher a novel role of PDPN-expressing LNSCs in the elimination of CD4⁺ TILs and propose a new target for tumor immunotherapy.