Tolerance induction in lymph nodes can be mediated by both haematopoietic cells (e.g. specific dendritic cells subsets) and by non-haematopoietic cells (e.g. lymph node stromal cells [LNSCs]) when they present peripheral tissue antigens to autoreactive T cells. LNSCs normally regulate T cell trafficking and survival and help to maintain peripheral tolerance by exerting immunosuppressive effects. However, whether autoimmunity can be associated with defective tolerogenic functions of LNSCs is unknown and studies aimed at characterising LNSCs in humans are lacking. We hypothesised that dysregulated T cell responses in pancreatic lymph nodes (PLNs) from donors with type 1 diabetes and from NOD mice may be associated with altered LNSC function. We analysed PLNs from donors with type 1 diabetes and NOD mice for LNSC distribution and phenotype using flow cytometry. We assessed the expression of tolerance-related genes in different subsets of LNSCs from human donors, as well as in a population of dendritic cells enriched in autoimmune regulator (AIRE)+ cells and identified as HLA-DRhigh CD45low. The relative frequency of different LNSC subsets was altered in both donors with type 1 diabetes and NOD mice, and both MHC class II and programmed death-ligand 1 (PD-L1) expression were upregulated in human type 1 diabetes. Tolerance-related genes showed similar expression profiles between mouse and human LNSCs at steady state but were generally upregulated in the context of human type 1 diabetes, while, at the same time, many such genes were downregulated in the AIRE-enriched dendritic cell population. Our study shows that LNSCs are substantially altered in type 1 diabetes, but, surprisingly, they exhibit an enhanced tolerogenic phenotype along with increased antigen-presenting potential, which may indicate an attempt to offset dendritic cell-related tolerogenic defects in tolerance. Thus, LNSCs could constitute alternative therapeutic targets in which to deliver antigens to help re-establish tolerance and prevent or treat type 1 diabetes. All data generated or analysed during this study are included in the published article (and its online supplementary files). Biomark gene expression data were deposited on the Mendeley repository at https://data.mendeley.com/datasets/d9rdzdmvyf/1 . Any other raw datasets are available from the corresponding author on reasonable request. No applicable resources were generated or analysed during the current study.