At the early stage of sepsis, it is clear that tumor necrosis factor alpha (TNF-α) will be excessively produced after mitogen-activated protein kinase is activated by lipopolysaccharide (LPS). TNF-α, which is an important early inflammatory cytokine, activates indirectly signal transducer and activator of transcription (STAT3) pathways. Activated STATs enter into the nucleus to take part in the gene expression induced by LPS. The present study was performed to explore the potential sites of STAT3 binding to TNF-α promoter. The purpose is to provide the theory basis of understanding molecular mechanism and intervention pathway of JAK/STAT signal pathway in the development of sepsis. In the present study, dose-dependent response of STAT3 on TNF-α expression was observed after Flag-STAT3 and full length TNF-α promoter reporter gene were co-transfected into COS-7 cells. The results indicated that TNF-α gene expression was enhanced along with increased doses of STAT3 with or without LPS. Flag-STAT3 (200 μg/L) and TNF-α promoter reporter gene of different length were co-transfected into COS-7 cells respectively, and LPS was added into cells 4 hours later. Compared to the controls, fold activity value of pTNF-α(95 bp) was found to be the raised 6.9-fold. Then, pTNF-α(70 bp), pTNF-α(75 bp), pTNF-α(80 bp) and pTNF-α(85 bp) deletion mutants were constructed and co-transfected with Flag-STAT3 (200 μg/L) to COS-7 cells induced by LPS. Data showed that fold activity value of pTNF-α(85 bp) was similar to that of pTNF-α(95 bp), and fold activity value of pTNF-α(80 bp) decreased to the control levels. 81 base and 82 base in the TNF-α promoter were mutated and then the activity results proved that the two sites were important to the TNF-α gene expression induced by STAT3. The potential binding site of STAT3 on TNF-α promoter was identified to be between 80 bp and 85 bp. Furthermore, the results of EMSA showed that it was wild probe tagged with γ-³²P of the TNF-α promoter 62～85 bp could bind to nucleic proein STAT3, but not mutant 62～85 bp probe. These data suggested that the effect of STAT3 on TNF-α gene expression did not depend on LPS, and the potential binding site of STAT3 on TNF-α promoter might be between 80 and 85 base radical fragment.