Oxidative stress is a known feature of calcific aortic valve disease (CAVD), but upstream activators of reactive oxygen species (ROS) are not well understood. We examined the role of inflammation in stimulating aortic valve endothelial cell (VEC) secretion of ROS. Using 3D culture, we determined that inflammatory cytokine TNF- α significantly increases VEC superoxide (223+/− 4% of control) and hydrogen peroxide (165 +/− 2% of control) within 5 minutes, blocked by the NOS inhibitor L-NAME, implicating eNOS uncoupling in ROS generation. Increased H2O2 production has long-term (48 hr) effects on VEC, causing increased ICAM-1, VCAM-1, and MMP-9, and decreased VE-cadherin and nitric oxide compared to untreated VEC. Similarly, we find that endothelial cells in calcified human aortic valves have elevated levels of superoxide, both near and distal to the primary nodules. Thus, the inflammatory phase of valve disease increases VEC oxidative stress, with long-term consequences including endothelial dysfunction. These results offer new paths for clinical intervention in early CAVD, such as using BH4 to promote eNOS coupling and reduce the downstream effects of inflammatory ROS. Funding: NIH 1R01HL110328–01