Increased expression of pituitary tumor-transforming gene 1 (PTTG1) occurs during mitosis-related sister chromatid segregation, and characterizes various tumor cells, including prostate cancer. Whereas the mechanism remains unclarified. Here, the PTTG1 levels in a prostate cancer cell line, PC3, were modulated by the expression of PTTG1 transgene or shRNA, showing that the PTTG1 levels affected the proliferation of prostate cancer cells, in vitro and in vivo. Moreover, a significant decrease in mothers against decapentaplegic homolog 3 (SMAD3), a key component of transforming growth factor β (TGFβ) signaling pathway, was induced by PTTG1 overexpression. Since SMAD3 is a ubiquitous cell-cycle inhibitor, our data suggest that PTTG1 may promote the proliferation of prostate cancer cells by inhibiting SMAD3-mediated TGFβ signaling. To identify a causal link, we expressed SMAD3 in PTTG1-overexpressing PC3 cells and found that SMAD3 expression inhibited the augmented cancer cell proliferation by PTTG1 overexpression. Furthermore, SMAD3 inhibition by short hairpin RNA (ShRNA) completely rescued the cancer cell proliferation in PTTG1 ShRNA-treated PC3 cells. Taken together, our data suggest that PTTG1 promotes the proliferation of prostate cancer cells via the inhibition of SMAD3. SMAD3 thus appears to be a novel therapeutic target for suppressing the growth of prostate cancer.