Human bone marrow derived multipotent mesenchymal stromal/stem cells (MSC) exhibit immunomodulatory properties. Previous studies have reported that both soluble and adhesion molecules produced by MSC are key contributing factors to their immunomodulatory effects. The contact dependent erythropoietin-producing hepatocellular (Eph) receptor tyrosine kinase family interact with their cognate ephrin ligands to regulate many biological processes, particularly thymocyte maturation and selection, T-cell transendothelial migration, activation, co-stimulation and proliferation. However, the contribution of Eph/ephrin interactions in mediating human MSC suppression of activated T-cells remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 are expressed by ex-vivo expanded human MSC, while the corresponding ligands, ephrin-B1 and EphB4, respectively, are highly expressed by human T-cells. To examine the role of EphB2/ephrin-B1 and ephrin-B2/EphB4 interactions in T-cell proliferation, we employed the mixed lymphocyte reaction (MLR) assay, where irradiated allogeneic lymphocytes were used to stimulate T-cell proliferation. Initial studies demonstrated that EphB2-Fc and ephrin-B2-Fc molecules could suppress T-cell proliferation in the MLR compared with human IgG treated controls. Whilst the addition of a third party MSC population demonstrated dramatic suppression of T-cell proliferation responses in the MLR, blocking the function of EphB2 or EphB4 receptors using inhibitor binding peptides significantly increased T-cell proliferation. Consistent with these observations, shRNA MSC knockdown lines for EphB2 or ephrin-B2 expression reduced their ability to inhibit T-cell proliferation. In addition, the expression of immunosuppressive factors, indoleamine 2,3-dioxygenase (IDO) and TGF-β1, expressed by MSC, was up-regulated following stimulation with EphB4 and ephrin-B1 compared with untreated controls. Conversely, key factors involved in T-cell activation and proliferation, IL-2, INF-γ, TNF-α and IL-17, were down-regulated by T-cells treated with EphB2 or ephrin-B2 compared with untreated controls. Our data suggest that inhibition of T-cell proliferation by human MSC is partly mediated through EphB2-induced ephrin-B1 reverse signalling or ephrin-B2-mediated EphB4 forward signalling. This work contributes to the current understanding of how MSC exert their immunosuppressive effects on activated T-cells, and may offer a unique therapeutic drug target to facilitate the regulation of T-cell populations in immune related conditions.