Background: Thrombin formation is a key feature in the activation of coagulation in pig xenograft recipients. As thrombin is known to activate endothelial and immune cells, we explored whether thrombin activation of pig endothelial cells (EC) was associated with an increased human T-cell response. Methods: α1,3-galactosyltransferase gene-knockout (GTKO) pig aortic EC (pAEC) were activated by porcine interferon-gamma (pIFNγ), human (h)IFN-γ, or thrombin. Swine leukocyte antigen (SLA) class I and class II expression were measured. Human peripheral blood mononuclear cells (PBMC) and CD4+ T-cell proliferation in response to activated pAEC, the effect of thrombin on pig CD80/CD86 mRNA, and the effect of thrombin inhibition by hirudin were evaluated. Results: After pAEC activation, SLA I expression did not change, and only pIFNγ upregulated SLA II expression. PBMC proliferation to pIFNγ- and thrombin-activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non-activated pAEC. CD4+ T-cell proliferation to pIFNγ- and thrombin-activated pAEC was significantly higher (P < 0.001 and P < 0.01) than to non-activated pAEC. Thrombin inhibition by hirudin reduced thrombin-induced upregulation of pAEC CD86 mRNA, and significantly reduced human PBMC proliferation to pAEC in comparison with thrombin alone (P < 0.05). Conclusions: Thrombin upregulates CD86 mRNA on pAEC, which is associated with increased human T-cell proliferation against pAEC. Hirudin reduces CD86 mRNA in thrombin-activated pAEC and is associated with downregulation of the human T-cell proliferative response. The transplantation of organs from GTKO pigs transgenic for human thrombomodulin, and/or endothelial protein C receptor, in addition to therapeutic regulation of thrombin activation may reduce the cellular response to a pig xenograft and thus reduce the need for intensive immunosuppressive therapy. © 2012 John Wiley & Sons A/S.