Thyroid peroxidase (TPO), a tissue-specific enzyme expressed in differentiated thyroid follicular cells, is a major antigen that has been linked to autoimmune thyroid disease. We have previously reported the functional expression of the lipopolysaccharide (LPS) receptor Toll-like receptor 4 on thyroid follicular cells. Here we investigated the effect of LPS in TPO expression and analyzed the mechanisms involved. We found a dose-dependent enhancement of TSH-induced TPO expression in response to LPS stimulation. EMSAs demonstrated that LPS treatment increased thyroid transcription factor-1 and -2 binding to the B and Z regions of TPO promoter, respectively. Moreover, LPS increased TSH-stimulated TPO promoter activity. Using bioinformatic analysis, we identified a conserved binding site for transcription nuclear factor-κB (NF-κB) in the TPO promoter. Chemical inhibition of NF-κB signaling and site-directed mutagenesis of the identified κB-cis-acting element abolished LPS stimulation. Furthermore, chromatin immunoprecipitation assays confirmed that TPO constitutes a novel NF-κB p65 subunit target gene in response to LPS. Additionally, our results indicate that p65 phosphorylation of serine 536 constitutes an essential step in the p65-dependent, LPS-induced transcriptional expression of TPO. In conclusion, here we demonstrated that LPS increases TPO expression, suggesting a novel mechanism involved in the regulation of a major thyroid autoantigen. Our results provide new insights into the potential effects of infectious processes on thyroid homeostasis.