Multipotential mesenchymal stem cells (MSC) exhibit immunomodulatory properties in vitro and in vivo. The contact dependent Eph/ephrin tyrosine kinase family regulate immune cell development and function, particularly thymocyte maturation and selection, T cell transendothelial migration, activation, co-stimulation and proliferation. However, the contribution of Eph/ephrin molecules in human MSC mediating activated T cell suppression remains to be determined. In the present study, we showed that EphB2 and ephrin-B2 were expressed by ex vivo expanded MSC, while their corresponding molecules, ephrin-B1 and EphB4 respectively, were highly expressed by T cell population, suggesting that EphB/ephrinB interactions may contribute to the MSC-mediated T cell suppression. To examine the importance of EphB2 and ephrin-B2 in T cell proliferation, we employed the mixed lymphocyte reaction assay, where irradiated allogeneic lymphocytes were used to stimulate T cell proliferation. We observed that EphB2 and ephrin-B2 could suppress T cell proliferation compared to untreated controls. In further experiments using EphB receptor binding peptides to block the function of EphB2 and ephrin-B2, we found that T cells exhibited increased proliferation. Consistent with these observations, experiments from shRNA MSC knockdown lines showed that inhibition of EphB2 and ephrin-B2 expression reduced MSC inhibitory function on T cell proliferation. Studies utilising signalling inhibitors showed that reverse ephrin-B1 signalling inhibited T cell proliferation by activating PI3Kinase pathway, while suppression mediated by EphB4 forward signalling were dependent on the Src kinase and Abl-dependent pathways. This study has identified that EphB/ephrinB interactions play a role in mediating human MSC inhibition of activated T-cells.