Abstract LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-κB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85α regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTENflox/flox/LysMCre mice (PTEN−/−), which have increased Akt activity. Analysis of LPS signaling in Pik3r1−/− and PTEN−/− cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IκBα or NF-κB nuclear translocation. LPS induction of TNF-α, IL-6, and tissue factor gene expression was increased in Pik3r1−/− peritoneal exudate cells and decreased in PTEN−/− peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1−/− bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85α subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.