Tumor necrosis factor alpha (TNF-alpha) is a major inflammatory mediator that exhibits actions leading to tissue destruction and hampering recovery from damage. At present, two antibodies against human TNF-alpha (hTNF-alpha) are available, which are widely used for the clinic treatment of certain inflammatory diseases. This work was undertaken to identify a novel functional epitope of hTNF-alpha. We performed screening peptide library against anti-hTNF-alpha antibodies, ELISA and competitive ELISA to obtain the epitope of hTNF-alpha. The key residues of the epitope were identified by means of combinatorial alanine scanning and site-specific mutagenesis. The N terminus (80-91 aa) of hTNF-alpha proved to be a novel epitope (YG1). The two amino acids of YG1, proline and valine, were identified as the key residues, which were important for hTNF-alpha biological function. Furthermore, the function of the epitope was addressed on an animal model of collagen-induced arthritis (CIA). CIA could be suppressed in an animal model by prevaccination with the derivative peptides of YG1. The antibodies of YG1 could also inhibit the cytotoxicity of hTNF-alpha. These results demonstrate that YG1 is a novel epitope associated with the biological function of hTNF-alpha and the antibodies against YG1 can inhibit the development of CIA in animal model, so it would be a potential target of new therapeutic antibodies.