Phosphoinositide-dependent kinase 1 (PDK-1) represents an important signaling component in the phosphatidylinositol 3-kinase (PI3K) pathway, which plays an essential role in controlling a coordinated innate immune response. Here, we show that mice with conditional disruption of PDK-1 specifically in myeloid lineage cells (PDK-1(Deltamyel) mice) show enhanced susceptibility to lipopolysaccharide (LPS)-induced septic shock accompanied by exaggerated liver failure. Furthermore, primary macrophages derived from PDK-1(Deltamyel) mice lack LPS- and Pam3CSK4-stimulated AKT activity but exhibit increased mRNA expression and release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Moreover, LPS- and Pam3CSK4-stimulated primary macrophages exhibit enhanced phosphorylation and degradation of IkappaBalpha. While immediate upstream Toll-like receptor 4 (TLR-4)-induced signaling, including IL-1 receptor (IL-1R)-associated protein kinase (IRAK) phosphorylation, is unaltered in the absence of PDK-1, macrophages from PDK-1(Deltamyel) mice exhibit prolonged ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF-6) in response to LPS stimulation. These experiments reveal a novel PDK-1-dependent negative feedback inhibition of TLR-induced NF-kappaB activation in macrophages in vivo.