Background: The major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated. Methods: We assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators (CD46, CD55 and CD59) and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus. Results: Recombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators (CD46, CD55 and CD59) increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs. Conclusion: Our data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.