Inflammatory cytokines have been linked to obesity-related insulin resistance. To investigate the effect of TNF-α, an inflammatory cytokine , on insulin action, C57BL/6J mice were treated with TNF-α for 7 days after which we examined the in vivo effects of TNF-α on glucose tolerance and insulin sensitivity with IV glucose tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, we analyzed the in vivo effect of TNF-α on several metabolism-related genes and adipocytokines implicated in the development of insulin resistance. TNF-α treatment resulted in markedly increased fasting blood glucose, insulin and free fatty acids (FFA) levels and reduced glucose tolerance. During the clamps, the rates insulin-stimulated whole body (G Rd) and skeletal muscle glucose uptake (MGU) and insulin’s ability to suppress hepatic glucose production (HGP) were decreased in TNF-α treated animals, indicating insulin resistance. In addition, both PPARγ and ATGL mRNA expression in adipose tissues as well as ATGL protein levels in plasma were downregulated. Moreover, adipose mRNA expression and plasma protein levels of adiponectin and visfatin were significantly down-regulated. We conclude that the alterations of PPARγ, ATGL, adiponectin and visfatin may contribute to the development of insulin resistance mediated by TNF-α.