Phosphoinositide-dependent protein kinase 1 (PDK1) plays a key role in the phosphoinositide 3-kinase (PI3K)-PDK1-Akt pathway that induces cell survival and cardiovascular protections through anti-apoptosis, vasodilation, anti-inflammation, and anti-oxidative stress activities. Although several reports have proposed the negative role of PDK1 in Toll-like receptor 4 (TLR4) signaling, the molecular mechanism is still unknown. Here we show that PDK1 inhibits tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) ubiquitination by interrupting the complex between transforming growth factor beta-activated kinase 1 (TAK1) and TAK1 binding protein 2 (TAB2), which negatively regulates TAK1 activity. The overexpression of PDK1 in 293/TLR4 cells resulted in suppressions of nuclear factor kappa B (NF-κB) activation and production of proinflammatory cytokines including interleukin (IL)-6 and TNF-α in response to lipopolysaccharide stimulation. Conversely, THP-1 human monocytes transiently cultured in low glucose medium displayed down-regulated PDK1 expression, and significantly enhanced TLR4-mediated signaling for the activation of NF-κB, demonstrating a negative role of PDK1. Biochemical studies revealed that PDK1 significantly interacted with TAK1, resulting in the inhibition of the association of TAB2 with TAK1, which led to the attenuation of TRAF6 ubiquitination. Moreover, PDK1-knockdown THP-1 cells displayed enhancement of downstream signals, activation of NF-κB, and increased production of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, which potentially led to the up-regulation of NF-κB-dependent genes in response to TLR4 stimulation. Collectively, the results demonstrate that PDK1 inhibits the formation of the TAK1-TAB2-TRAF6 complex and leads to the inhibition of TRAF6 ubiquitination, which negatively regulates the TLR4-mediated signaling for NF-κB activation.