Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-beta (TGF-beta)-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-beta signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-beta signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-beta-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-beta/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.