Phosphorylated derivatives of the lipid phosphatidylinositol are known to play critical roles in insulin response. Phosphatidylinositol 5-phosphate 4-kinases convert phosphatidylinositol 5-phosphate to phosphatidylinositol 4,5-bis-phosphate. To understand the physiological role of these kinases, we generated mice that do not express phosphatidylinositol 5-phosphate 4-kinase β. These mice are hypersensitive to insulin and have reduced body weights compared to wild-type littermates. While adult male mice lacking phosphatidylinositol 5-phosphate 4-kinase β have significantly less body fat than wild-type littermates, female mice lacking phosphatidylinositol 5-phosphate 4-kinase β have increased insulin sensitivity in the presence of normal adiposity. Furthermore, in vivo insulin-induced activation of the protein kinase Akt is enhanced in skeletal muscle and liver from mice lacking phosphatidylinositol 5-phosphate 4-kinase β. These results indicate that phosphatidylinositol 5-phosphate 4-kinase β plays a role in determining insulin sensitivity and adiposity in vivo and suggest that inhibitors of this enzyme may be useful in the treatment of type 2 diabetes.