Smads are important intracellular effectors in signaling pathways of the transforming growth factor-beta (TGF-beta). Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. SMAD4 is an important tumor suppressor gene. Smad4 has been shown to be constitutively phosphorylated, but the kinase that performs this phosphorylation is unknown. In this study, Smad4 was identified to interact with Nemo-like kinase (NLK) by a yeast two-hybrid system, and this interaction was confirmed in vitro and in vivo. Furthermore, the linker sequence of Smad4 is sufficient for this specific interaction. NLK is a conserved Ser/Thr kinase. Using in vitro kinase assays, we identified that threonine 9 (Thr9) and Serine 138 (Ser138) within the N-terminal Mad homology1 (MH1) domain of Smad4 could be phosphorylated by NLK. Our research suggests that NLK may play a novel role in the regulatory of Smad4 through phosphorylation.