Insulin regulates hepatic VLDL production by activation of phosphatidylinositide 3-kinase (PI 3-kinase) which decreases apo B available for lipid assembly. The current study evaluated the dependence of the VLDL apo B pathway on PI 3-kinase activity in vivo. VLDL production was examined in B100 only, apobec-1^-/- mice, using the Triton WR 1339 method. Glucose injection suppressed VLDL triglyceride production by 28% in male and by 32% in female mice compared with saline-injected controls. When wortmannin was injected to inhibit PI 3-kinase, VLDL triglyceride production was increased by 52% in males, and by 89% in females, and VLDL B100 levels paralleled triglyceride changes. Pulse-chase experiments in primary mouse hepatocytes showed that wortmannin increased net freshly synthesized B100 availability by more than 35%. To test whether physiologic insulin resistance produced equivalent effects to wortmannin, we studied male apobec-1^-/- mice who became hyperlipidemic upon feeding a fructose-enriched diet. Fructose-fed apobec-1^-/- mice had significantly higher VLDL triglyceride and B100 production rates compared with chow-fed mice and rates were refractile to glucose or wortmannin. Hepatic VLDL triglyceride and B100 production in wortmannin-injected chow-fed mice equaled that observed in fructose-fed mice. Together, results suggest in vivo and in vitro that wortmannin sensitive PI 3-kinases maintain a basal level of VLDL suppression which is sensitive to changes in activation, and which can increase VLDL production when PI 3-kinase is inhibited to levels similar to those induced by insulin resistance.