TNF- plays an important role in obesity-linked insulin resistance and diabetes mellitus by activating at least two serine kinases capable of promoting negative regulation of key elements of the insulin signaling pathway. Pharmacological inhibition of TNF- is currently in use for the treatment of rheumatoid and psoriatic arthritis and some case reports have shown clinical improvement of diabetes in patients treated with the TNF- blocking monoclonal antibody, infliximab. The objective of this study was to evaluate the effect of infliximab on glucose homeostasis and insulin signal transduction in an animal model of diabetes. Diabetes was induced in Swiss mice by a fat-rich diet. Glucose and insulin homeostasis were evaluated by glucose and insulin tolerance tests, and by the hyperinsulinemic-euglycemic clamp. Signal transduction was evaluated by immunoprecipitation and immunobloting assays. Short-term treatment with infliximab rapidly reduced blood glucose and insulin levels and glucose and insulin areas under curve during a glucose tolerance test. Furthermore, infliximab increased the glucose decay constant during an insulin tolerance test and promoted a significant increase in glucose infusion rate during a hyperinsulinemic-euglycemic clamp. In addition, the clinical outcomes were accompanied by improved insulin signal transduction in muscle, liver and hypothalamus, as determined by the evaluation of insulin-induced IR, IRS-1 and IRS-2 tyrosine phosphorylation and Akt and FOXO1 serine phosphorylation. Thus, pharmacological inhibition of TNF- may be an attractive approach to treat severely insulin resistant patients with type 2 diabetes mellitus.