Apoptosis and epithelial-to-mesenchymal transdifferentiation or transition (EMT) are crucial for normal development and body homeostasis. The alterations of these events are closely related to some pathologic processes, such as tumor formation and metastasis, fibrotic diseases of liver and kidney, and abnormal development of embryos. The mechanism that underlies the simultaneously occurring apoptosis and EMT induced by transforming growth factor-β (TGF-β) has not been well studied. In this report, we investigated the potential mechanism that underlies TGF-β1–induced apoptosis and EMT. TGF-β1–induced apoptosis and EMT were associated with the activation of protein kinase A (PKA) and signal transducers and activators of transcription 3 (STAT3). Inhibition of PKA by specific PKA inhibitor H89 or by PKA inhibitor peptide blocked STAT3 activation and suppressed TGF-β1–induced apoptosis and EMT. Furthermore, overexpression of a phosphorylation-deficient form of STAT3, but not wild-type STAT3, produced an inhibitory effect on TGF-β1–induced apoptosis and EMT. The results indicate that PKA is an upstream regulator for TGF-β1–induced STAT3 activation and plays an important role in TGF-β1–mediated apoptosis and EMT. These studies provided a new insight into the signaling mechanism underlying the apoptosis and EMT, which could be of importance in understanding some related physiologic and pathologic processes. (Cancer Res 2006; 66(17): 8617-24)