Although SMAD3 signaling has been suggested to play a role in the metastasis of various cancers, its possible involvement as well as the underlying mechanism in the pathogenesis in prostate cancer remains unclear. Here, we found that the MMP9 level, an indicator of the invasiveness of cancer cells, negatively correlates with the activity of phosphorylated SMAD3 levels in the prostate cancer patients. Moreover, the phosphorylated SMAD3 also appeared to regulate the MMP9 level in a prostate cancer cell line, PC3. Augmented phosphorylated SMAD3 inhibited MMP9 and invasiveness of PC3 cells, while inhibition of phosphorylated SMAD3 activated MMP9 and promoted PC3 cell invasiveness. Furthermore, forced MMP9 inhibition abolished the effect of phosphorylated SMAD3 on the invasiveness of PC3 cells, while forced MMP9 activation abolished the effect of phosphorylated SMAD3 on the invasiveness of PC3 cells. Taken together, our data suggest the possibility of the existence of a unique signaling cascade in which SMAD3 signaling regulates MMP9 during cancer metastasis.