Obesity is now considered a chronic, low grade, inflammatory disease and macrophages have been shown to play a crucial role in the setting of insulin resistance. Macrophages, particularly M1 polarized, pro-inflammatory macrophages, accumulate in the adipose tissue and other insulin-responsive organs and contribute to insulin resistance via the secretion of pro-inflammatory cytokines which disrupt insulin action. The precise mechanisms regulating the inflammatory response in macrophages are unclear, however it has been suggested that the transcription factor FoxO1 may mediate the production of inflammatory cytokines in macrophages. To investigate the precise role of FoxO1 in macrophages in the etiology of insulin resistance, we have specifically deleted FoxO1 from macrophages of mice using Cre-Lox technology. In vitro, we show that the deletion of FoxO1 from cultured bone marrow derived macrophages blunts the mRNA expression of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, but increases the mRNA expression of anti-inflammatory cytokine IL-10. When fed a high fat diet (HFD), mice lacking FoxO1 in macrophages (MacFoxO1-/-) gain less weight compared with WT mice, as a result of gaining markedly less body fat (WT 12.4 +/- 0.96 vs KO 8.2 +/- 0.65 g, p<0.005). In addition, MacFoxO1-/- mice display reduced hepatic steatosis when fed the HFD. Moreover, MacFoxO1-/- mice are completely protected against the deleterious effects of high fat feeding on glucose tolerance as measured by the incremental area under the glucose curve during an intraperitoneal glucose tolerance test (WT 472.1 +/- 83 vs KO 272.9 +/- 31 mmol glucose, p<0.05). Finally, MacFoxO1-/- mice showed a tendency to have less macrophages in adipose tissue as measured by staining for the macrophage marker F4/80, in adipose tissue sections. Together, these data suggest that FoxO1 is an important transcription factor mediating inflammation in macrophages, and contributes to the pathogenesis in obesity-induced insulin resistance.