Transforming growth factor (TGF)-1 has been reported to cause endothelial cell (EC) apoptosis and promotes EC survival. In this study, we investigated the effect of TGF-1 on apoptosis of cultured bovine pulmonary artery endothelial cells (BPAEC) induced by multiple stimuli. We demonstrated that TGF-1 protected against apoptosis of BPAEC induced by either serum deprivation or the VEGF receptor inhibitor, SU5416, but not by UV light exposure or TNF. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9; an effect that was abolished by TGF-1. We further demonstrated that both serum deprivation and inhibition of VEGF receptors significantly decreased protein level of Bcl-2; effects that were also abrogated by TGF-1. In addition, the baseline level of Bcl-2 was enhanced by TGF-1 and reduced by inhibition of ALK5, a TGF-1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of BPAEC. These results suggest that TGF-1 signaling is critical for maintenance of BPAEC survival. Finally, we demonstrated that the protective effects of TGF-β1 on BPAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. TGF-1 activated both SMAD2 and SMAD1/5; effects that were abolished by ALK5 inhibition. These results suggest that TGF-1 protects against BPAEC apoptosis possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of mitochondrial-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.