The TAK1-NLK cascade is a mitogen-activated protein kinase-related pathway that plays an inhibitory role in canonical Wnt/beta-catenin signaling through regulating the LEF1/TCF family transcriptional factors. TAB2 (TAK1-binding protein 2) is a putative TAK1 interacting protein that is involved in the regulation of TAK1. Here, we found that TAB2 could directly interact with NLK and function as a scaffold protein to facilitate the interaction between TAK1 and NLK. Knocking down TAB2 using small interfering RNA abolished the interaction of TAK1 with NLK in mammalian cells. The intermediate region (residues 292-417) of TAB2 was mapped for its binding to NLK. TAB2-DeltaM, a TAB2 mutant lacking this region, showed a lower affinity for NLK and became defective in its scaffolding function. In addition, TAB2, but not TAB2-DeltaM, mediated TAK1-dependent activation of NLK and LEF1 polyubiquitylation, resulting in the inhibition of canonical Wnt signaling. Moreover, Wnt3a stimulation led to an increase in the interaction of TAB2 with NLK and the formation of a TAK1.TAB2.NLK complex, suggesting that this TAK1-TAB2-NLK pathway may constitute a negative feedback mechanism for canonical Wnt signaling.