The adaptive response of endothelial cells to hypoxia involves a substantial remodeling of extracellular matrix (ECM). In endothelial cells hypoxia up-regulates lysyl oxidase (LOX), a key enzyme in ECM assembly, relevant to vascular homeostasis. However, the mechanism underlying this response has not been established. Hypoxia up-regulated LOX expression in endothelial cells (HUVEC and BAEC) and concomitantly increased LOX enzymatic activity. This effect was independent of autocrine factors released by hypoxic cells and relies on a transcriptional mechanism. Both mTOR blockade and HIF-1alpha knockdown slightly prevented LOX up-regulation by hypoxia, suggesting that HIF-1alpha is only partially responsible for this effect. In fact, serial promoter deletion and mutagenesis studies indicated a limited contribution of the previously described hypoxia response element (-75 bp). Interestingly, Smad over-expression further increased LOX transcriptional activity in endothelial cells exposed to hypoxia. Moreover, the increase in LOX expression triggered by hypoxia was significantly reduced by reactive oxygen species (ROS) inhibitors. Thus, our data support a role of Smad signaling and ROS in the up-regulation of LOX by hypoxia in endothelial cells.