Inhibition of the CD40–CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti-CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40-driven inflammatory disorders. Blocking tumour necrosis factor receptor-associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II+ cells diminishes inflammation. However, CD40–TRAF6 blockade may cause immunosuppression. We examined the role of CD40–TRAF2,3 and CD40–TRAF6 signalling in the development of pro-inflammatory responses in human non-haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild-type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non-haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154-induced up-regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), monocyte chemotactic protein 1 (MCP-1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM-1 up-regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP-1 and tissue factor up-regulation in these cells. Treatment of endothelial and smooth muscle cells with cell-permeable peptides that block CD40–TRAF2,3 or CD40–TRAF6 signalling impaired pro-inflammatory responses. In contrast, while the CD40–TRAF2,3 blocking peptide did not reduce CD154-induced dendritic cell maturation, the CD40–TRAF6 blocking peptide impaired this response. Hence, preventing CD40–TRAF2,3 or CD40–TRAF6 interaction inhibits pro-inflammatory responses in human non-haematopoietic cells. In contrast to inhibition of CD40–TRAF6 signalling, inhibition of CD40–TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40–TRAF2,3 signalling may control CD40–CD154-dependent inflammatory disorders.