The development of intestinal permeability and the penetration of microbial products are key factors associated with the onset of metabolic disease. However, the mechanisms underlying this remain unclear. Here we show that, unlike liver or adipose tissue, high fat diet (HFD)/obesity in mice does not cause monocyte/macrophage infiltration into the intestine or pro-inflammatory changes in gene expression. Rather HFD causes depletion of intestinal eosinophils associated with the onset of intestinal permeability. Intestinal eosinophil numbers were restored by returning HFD fed mice to normal chow and were unchanged in leptin-deficient (Ob/Ob) mice, indicating that eosinophil depletion is caused specifically by a high fat diet and not obesity per se. Analysis of different aspects of intestinal permeability in HFD fed and Ob/Ob mice shows an association between eosinophil depletion and ileal paracelullar permeability, as well as leakage of albumin into the feces, but not overall permeability to FITC dextran. These findings provide the first evidence that a high fat diet causes intestinal eosinophil depletion, rather than inflammation, which may contribute to defective barrier integrity and the onset of metabolic disease.