Abnormal and prolonged inflammatory reaction is seen in a wide variety of disorders, and high level of Tumor Necrosis Factor alpha (TNF-α) has been linked to these disorders. Therefore, modulation of TNF-α expression is important in the regulation of inflammatory disorders. In our previous study, we have shown that a transcription factor LPS-induced TNF factor (LITAF) significantly induces TNF-α production. Furthermore, we found that p53 and its synthetic peptide 162-motif specifically downregulate LITAF/TNF-α gene expression in human cells in vitro. Thus, in the present study, the role of p53 in regulating TNF-α-mediated inflammation was investigated. Our data showed that a synthetic peptide, named 162-motif, corresponding to this region functions independently from p53 to cause a significant suppression of TNF-α gene expression in mouse primary macrophages. The 162-motif, when delivered into cells and organs, reduces serum TNF-α level in mice and prevents TNF-α-induced lung lesions and endotoxic shock. Our findings highlight the regulation of LITAF/TNF-α by p53 and its short peptide 162-motif. These in vitro and in vivo observations serve to pave the way for pharmacotherapeutic approaches in the treatment of inflammatory diseases.