Salt-inducible kinases (SIKs) are a family of related serine-threonine kinases and are involved in controlling various metabolisms such as liver glucose homeostasis, hepatic lipogenesis, steroidogenesis, and adipogenesis. Here we investigated the regulatory role of SIK proteins in Toll-like receptor 4 (TLR4)-mediated signaling. Overexpression of SIK1 and SIK3, but not SIK2, significantly inhibited nuclear factor-κB activity in response to lipopolysaccharide stimulation and affected the expression of proinflammatory cytokines. In contrast, both SIK1(KD) and SIK3(KD) Raw 264.7 cells exhibit dramatic elevations of nuclear factor-κB activation and activations of downstream signaling molecules, such as TGF-β-activated kinase 1, p38, and c-Jun N-terminal kinase, in response to TLR4 stimulation, indicating that SIK1 and SIK3 are negatively involved in the TLR4-mediated signaling. Through biochemical studies, we found that SIK1 and SIK3 interact with TGF-β-activated kinase 1-binding protein 2 (TAB2), and interrupt the functional complex of TAB2-TNF receptor-associated factor 6 (TRAF6). Interestingly, the molecular interruption is induced to suppress the ubiquitination of TRAF6 in response to TLR4 stimulation. These result suggest that SIK1 and SIK3 negatively regulate TLR4-mediated signaling through the interruption of TAB2-TRAF6 complex and thereby the inhibition of ubiquitination of TRAF6. The present findings can be useful for a better understanding of multilevel interactions between the metabolic and immune systems.