<p>Most cells produce latent transforming growth factor-beta 1 (TGF-β1), but only very few activate the cytokine via cell type-specific mechanisms. TGF-β1 favors cancer progression by suppressing anti-tumor T cell responses. Which cells produce this immunosuppressive TGF-β1 in human tumors is unknown. Putative sources include cells expressing the glycoprotein A repetitions predominant (GARP) protein, comprising mostly activated regulatory T cells (Tregs) (GARP⁺FOXP3⁺ cells) and blood endothelial cells (BECs). We performed multiplexed immunohistofluorescence and computerized image analyses on 186 tumor samples from 5 cancer types (colorectal, urothelial, lung and breast primary carcinomas and melanoma metastases), compared to patient-matched adjacent non-cancerous tissues. GARP⁺ Tregs were present in 29–75% of the various types of tumor samples. Their proportion was higher in tumors than non-cancerous tissues but unexpectedly it did not correlate with that of tumor-infiltrating T lymphocytes (TILs). The density of blood vessels was similar across samples, with more than half expressing GARP. The proportion of cells undergoing TGF-β1 signaling, which express the phosphorylated form of mothers against decapentaplegic homolog 2 (pSMAD2), was approximately twice as high in tumors compared to non-cancerous tissues. In most tumor types, pSMAD2⁺ TILs were twice closer to the nearest FOXP3⁺ cell than after random repositioning, at a distance (~ 70 µm) consistent with short-range paracrine TGF-β1 signaling. In contrast, pSMAD2⁺ non-T cells and pSMAD2⁻ TILs were not closer to FOXP3⁺ cells, neither were pSMAD2⁺ cells (TILs and others) to BECs. We conclude that, in human tumors, GARP-expressing Tregs rather than BECs appear to represent a source of TGF-β1 suppressing nearby TILs. This local immunosuppression could be blocked with anti-GARP:TGF-β1 antibodies, particularly to treat patients with tumors heavily infiltrated by GARP-expressing Tregs.</p>