We have previously shown that TGF-1 protected against main pulmonary artery endothelial cell (PAEC) apoptosis induced by serum deprivation and VEGF receptor blockade through a mechanism associated with ALK5-mediated Bcl-2 upregulation. In the current study, we investigated the effect of TGF-1 on pulmonary microvascular endothelial cell (PMVEC) apoptosis. We found that, in contrast to the results seen in conduit PAEC, TGF-1 caused apoptosis of PMVEC; an effect that was also dependent upon ALK5 activity. We noted that non-SMAD signaling pathways did not play a role in TGF-1-induced apoptosis. Both SMAD2 and SMAD1/5 were activated upon exposure to TGF-1. TGF-1-induced activation of SMAD2, but not SMAD1/5, was abolished by ALK5 inhibition; an effect associated with prevention of TGF-1-induced apoptosis. These results suggest that SMAD2 is important in TGF-1-induced apoptosis of PMVEC. While caspase-12 activity was not altered, caspase-8 was activated by TGF-1; an effect which correlated with a reduction of cFLIP protein levels. Additionally, TGF-1 decreased Bcl-2 protein levels and induced cytochrome c cytosolic redistribution. These results suggest that TGF-1 caused apoptosis of PMVEC likely through both caspase-8-dependent extrinsic pathway and mitochondria-mediated intrinsic pathway. We noted that inhibition of ALK5 attenuated serum deprivation-induced apoptosis; an effect correlated with increased expression and activation of CREB and its potential target genes, Bcl-2 and cFLIP. These results suggest that CREB may be important in mediating apoptosis resistance of PMVEC upon ALK5 inhibition perhaps through upregulation of Bcl-2 and cFLIP. Finally, we noted that SMAD1/5 were activated upon ALK5 inhibition in the presence of low dose of TGF-1; an effect associated with enhanced endothelial proliferation. We speculate that imbalance of ALK1 and ALK5 may contribute to the development of pulmonary artery hypertension.