Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, and survival in most cell types. Recently, we characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1), which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about the role of PIK3IP1 in normal and neoplastic growth in vivo. Proper liver function and development depend on intact PI3K signal transduction; when dysregulated, the PI3K pathway is linked to the development of liver cancer. To begin to dissect the contribution of PIK3IP1 to hepatic PI3K signaling in vivo and to liver tumorigenesis in particular, we formulated the following hypothesis: because PIK3IP1 down-regulates PI3K signaling and uncontrolled PI3K signaling is associated with liver cancer, then PIK3IP1-mediated down-regulation of the PI3K pathway should inhibit hepatocellular carcinoma (HCC) development. To test this idea, we generated transgenic mice overexpressing PIK3IP1 in hepatocytes in a mouse strain prone to develop HCC. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility, and survival compared with controls. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics compared with controls. We also observed that human HCC expressed less PIK3IP1 protein than adjacent matched liver tissue. Our data show that PIK3IP1 is an important regulator of PI3K in vivo, and its dysregulation can contribute to liver carcinogenesis. [Cancer Res 2008;68(14):5591–8]