Runx2 is a bone-specific transcription factor that plays a critical role in bone development, postnatal bone formation, and chondrocyte maturation. The protein levels of Runx2 are regulated by the ubiquitin-proteasome pathway. In previous studies we discovered that E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1) induces Runx2 degradation in a ubiquitin- proteasome-dependent manner, and Smurf1 plays an important role in osteoblast function and bone formation. In the present studies we investigated the molecular mechanism of Smurf1-induced Runx2 degradation. Smurf1 interacts with the PY motif of substrate proteins, and a PY motif has been identified in the C terminus of the Runx2 protein. To determine whether Smurf1 induces Runx2 degradation through the interaction with the PY motif of Runx2, we created amutant Runx2 with a PYmotif deletion and found that Smurf1 retained some of its ability to induce the degradation of the mutant Runx2, suggesting that Smurf1 could induce Runx2 degradation through an indirect mechanism. Smurf1 has been shown to interact with Smads 1, 5, 6, and 7, and Smads 1 and 5 also interact with Runx2. In the present studies we found that Smads 1 and 5 had no effect on Smurf1-induced Runx2 degradation. Although Smads 6 and 7 bind Smurf1, it is not known if Smads 6 or 7 interacts with Runx2 and mediate Runx2 degradation. We performed immunoprecipitation assays and found that Smad6 but not Smad7 interacts with Runx2. Smad6 enhances Smurf1-induced Runx2 degradation in an ubiquitin-proteasome-dependent manner. These results demonstrate that in addition to its interaction with the PY motif of Runx2, Smurf1 induces Runx2 degradation in a Smad6-dependent manner. Smurf1-induced Runx2 degradation serves as a negative regulatory mechanism for the BMP-Smad-Runx2 signaling pathway.