Omentin is a GPI-anchored 38-kDa secreted insulin-sensitizing glycoprotein that is expressed in the mesothelial cells of the stromal vascular fraction (SVF) of visceral adipose tissue. Mesothelial cells expressing omentin are part of the innate immune system and can contribute to the inflammatory state of visceral adipose tissue. Therefore, it is important to determine the role that omentin plays in the regulation of chronic inflammation. Primary cultures of human adipocyte mesothelial cells were isolated by biopsy trypsinization and Immunofluorescence microscopy was used to determine the purity of primary HAMC cultures. To determine if chronic inflammation is the cause of decreased omentin expression in obesity, HAMCs were treated with TNF-alpha, a pro-inflammatory cytokine, for 24 hours to simulate chronic exposure. Omentin gene expression was down-regulated by TNF-alpha significantly (39% decrease) in serum-free chronic conditions. The protein expression of omentin was also significantly decreased (31%) in serum containing conditions. To demonstrate that omentin's insulin-sensitizing activity may be mediated by anti-inflammatory actions, inflammation-dependent NF-kB luciferase reporter assays were performed in 3T3-L1 adipocytes and human adipose mesothelial cells treated with omentin and/or TNF-alpha. Omentin decreased pro-inflammatory gene transcription in 3T3-L1 adipocytes (85% decrease) and in HAMCs (68% decrease). In order to ascertain the role that omentin plays in the regulation cytokine secretion and what would result if omentin were not present, lentivirus containing omentin-specific shRNAs were generated. A complete knockdown was not achieved, but stimulation with LPS significantly increased (1.73%) the amount of TNF-alpha secretion in mesothelial cells suggesting that even partially reduced omentin protein expression can have a profound impact on the mesothelial inflammatory response. In conclusion, omentin plays a role in the anti-inflammatory response, but can also be negatively regulated by excessive and chronic pro-inflammatory cytokines.