Abstract Body: Atherogenic endothelial activation is influenced by the local microenvironment, including matrix composition and disturbed blood flow, as well as soluble proinflammatory stimuli such as oxidized LDL (oxLDL). While ER stress is known to contribute to atherogenic endothelial activation, the mechanisms triggering ER stress in atheroprone regions have remained unclear. Enhanced deposition of fibronectin (FN) in the subendothelial basement membrane at atheroprone sites has been observed, and this study shows that disturbed flow and oxLDL induce ER stress only in endothelial cells adhered to FN, not basement membrane. Prevention of integrin activation (via talin1 L325R mutation) inhibited ER stress induced by flow and oxLDL but did not affect tunicamycin-induced ER stress. ER stress was further induced through activation of FN-binding integrins (α5β1, αvβ3) using specific antibodies or CHAMP peptides. Blocking ER stress with TUDCA reduced flow- and oxLDL-induced VCAM-1 expression on FN and was linked to decreased JNK activation, though NF-κB activation remained unaffected. Inhibition of JNK target c-Jun also prevented flow- and oxLDL-induced VCAM-1 expression. Distinct roles for FN-binding integrins in ER stress were found, with αvβ3 required for flow-induced ER stress and α5β1 mediating oxLDL-induced ER stress. Endothelial cell-specific knockout (iEC) of α5 or αv integrins in mice resulted in reduced ER stress markers at atheroprone sites. Mechanistically, α5-mediated c-ABL and αv-mediated PAK signaling were identified as pathways driving oxLDL- and flow-induced ER stress, respectively. Inhibition of c-ABL and PAK blunted ER stress responses to oxLDL and flow. These findings provide the first evidence that matrix composition plays a critical regulatory role in ER stress, with significant implications for atherogenic endothelial activation.