Phosphatidylinositol 3-kinase (PI3K) plays a central role in insulin signaling, glucose metabolism, cell growth, cell development, and apoptosis. A heterozygous missense mutation (R649W) in the p85α regulatory subunit gene of PI3K (PIK3R1) has been identified in patients with SHORT (Short stature, Hyperextensibility/Hernia, Ocular depression, Rieger anomaly, and Teething delay) syndrome, a disorder characterized by postnatal growth retardation, insulin resistance, and partial lipodystrophy. Knock-in mice with the same heterozygous mutation mirror the human phenotype. In this study, we show that Pik3r1 R649W knock-in mice fed a high-fat diet (HFD) have reduced weight gain and adipose accumulation. This is accompanied by reduced expression of several genes involved in lipid metabolism. Interestingly, despite the lower level of adiposity, the HFD knock-in mice are more hyperglycemic and more insulin-resistant than HFD-fed control mice. Likewise, when crossed with genetically obese ob/ob mice, the ob/ob mice carrying the heterozygous R649W mutation were protected from obesity and hepatic steatosis but developed a severe diabetic state. Together, our data demonstrate a central role of PI3K in development of obesity and fatty liver disease, separating these effects from the role of PI3K in insulin resistance and the resultant hyperglycemia.