Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85 regulatory and a p110 catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor, wortmannin, and small-interfering RNA (siRNA) targeting the p85 and p110 subunits to determine if total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin, and selective inhibition of p85 or p110 with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest timepoints. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85 or p110. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.