TNFalpha is a key factor in the pathogenesis of rheumatoid arthritis. To investigate whether heterologous TNFalpha gene vaccination could induce anti-TNFalpha antibodies via cross-reaction and prevent the inflammatory arthritis, we constructed two plasmids by inserting a full-length cDNA of human TNFalpha into a secreted vector (pSecTag-TNFalpha) and a non-secreted vector (pTARGE-TNFalpha), respectively. Administering either plasmid to collagen-induced arthritis (CIA) mice reduced paw swelling and synovium-infiltrating inflammatory cells. This reduction was accompanied by down-regulated TNFalpha in sera and joints. The spleen cells from treated CIA mice displayed decreased IFN-gamma mRNA levels and matrix metalloproteinase-9 bioactivity in comparison with those from CIA control. Furthermore, both spontaneous and collagen-specific proliferation of the lymphocytes was significantly decreased after treatment. Administration of plasmids led to an elicited production of antibodies to both human and mouse TNFalpha. These results suggest that human TNFalpha gene vaccination prevents CIA in mice likely by inducing cross-reactive antibodies against TNFalpha, and that heterologous gene vaccination might provide an effective therapeutic strategy to battle TNFalpha mediated diseases.