SMAD-4 plays an important role in tumor suppression though controversies still exist regarding its behavior in carcinogenesis and its relationship with the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which is regarded as a key controller of cell cycle progression and cell growth. The role of SMAD-4 on PTEN expression was investigated through TGF-β signaling in 293T cells and in malignant gastric carcinoma MGC-803 cells. Results showed that SMAD-4 and TGF-β enhanced the expression of PTEN in 293T cells, while they suppressed PTEN expression in MGC-803 cells. However, this suppression was relieved upon the inhibition of RAS/ERK pathway. Moreover, the maximum expression of PTEN was achieved by the cooperation of SMAD-4 with TGF-β when SMAD-4 was translocated into the nucleus. Enhancement of early apoptosis of about three folds was achieved with this cooperation, compared with the action of TGF-β alone in MGC-803 cells. These findings shed light on the role of SMAD-4 as a co-Smad protein in TGF-β protein-signaling and in PTEN regulation.