Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.