Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/ signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITPα in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITPα selectively ablated for PtdIns binding activity (PitpαT59D), as the sole source of PITPα, exhibit phenotypes that recapitulate those of authentic PITPα nullizygotes. Analyses of mice with graded reductions in PITPα activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITPα protein levels are strongly reduced (≥90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITPα expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitpα0/0 neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITPα in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITPα-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia. Copyright © 2007 by the American Society for Biochemistry and Molecular Biology, Inc.