TNFR-associated factor (TRAF)6 integrates signals from multiple cell surface receptors for the activation of NFkB. However, the mechanism underlying LPS-induced TRAF6 signaling remains unclear. We report that cullin-5 (Cul-5), a cullin family scaffold protein, binds to TRAF6 and promotes TRAF6 polyubiquitination at Lys63 in response to LPS stimulation. A direct interaction between the C-terminal domain of Cul-5 and the TRAF-C domain of TRAF6 facilitates polyubiquitination of TRAF6. Hemizygous Cul-5 knockout is associated with improved survival of mice following LPS challenge and significant delays in the phosphorylation of p65/RelA, ERK, JNK, and p38 MAPKs in LPS-stimulated macrophages, along with a marked decrease in NF-κB activation. These findings identify Cul-5 as a signaling component that connects an LPS-activated TLR4-MyD88 complex to TRAF6 for efficient activation of NF-κB.