Abstract Body (Do not enter title and authors here): Atherogenic endothelial activation arises from both the local arterial microenvironment—characterized by altered extracellular matrix composition and disturbed blood flow—and soluble proinflammatory stimuli such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, enhances the proinflammatory effects of oxLDL and disturbed flow. While endoplasmic reticulum (ER) stress is known to contribute to vascular dysfunction, its regulation by matrix composition remains unclear. Here, we show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, but not in those adhered to basement membrane proteins. This matrix-specific ER stress response requires integrin activation, as endothelial cells expressing an integrin activation–deficient mutant (talin1 L325R) fail to activate ER stress in response to disturbed flow and oxLDL, while direct stimulation of integrins using CHAMP peptides is sufficient to induce ER stress. Silencing fibronectin-binding integrins (α5, αv) using siRNA blocks ER stress induction in vitro, and endothelial-specific deletion of α5 or αv reduces ER stress at atheroprone regions in vivo. Mechanistically, integrin-dependent ER stress is not associated with increased protein synthesis, unfolded protein accumulation, or superoxide production. Scavenging superoxide with TEMPOL does not alleviate ER stress. However, pharmacological inhibition of ER stress using TUDCA suppresses proinflammatory and metabolic gene expression (bulk RNA-seq), without affecting NF-κB activation. Instead, TUDCA prevents activation of the JNK–c-Jun signaling axis, which we show to be essential for proinflammatory gene induction. Blocking this pathway using a JNK inhibitor (SP600125) or dominant-negative c-Jun (TAM67) abrogates inflammatory gene expression following oxLDL or disturbed flow. Together, these findings identify a novel mechanism by which fibronectin–integrin signaling promotes ER stress in response to mechanical and metabolic stressors, amplifying endothelial inflammation through JNK–c-Jun signaling and contributing to atherogenesis.