Dyslipidemia is a primary risk factor for cardiovascular disease, but the specific mechanisms that determine the localization of atherosclerotic plaques in arteries are not well defined. Triglyceride-rich lipoproteins (TGRL) isolated from human plasma after a high-fat meal modulate TNF-α-induced VCAM-1 expression in cultured human aortic endothelial cells (HAECs) via an interferon regulatory factor (IRF)-1-dependent transcriptional mechanism. We examined whether fluid shear stress acts as a mediator of IRF-1-dependent VCAM-1 expression in response to cytokine and dietary lipids. IRF-1 and VCAM-1 were examined by immunofluorescence in TNF-α-stimulated HAEC monolayers exposed to TGRL and a linear gradient of shear stress ranging from 0 to 16 dyn/cm² in a microfluidic device. Shear stress alone modulated TNF-α-induced VCAM-1 expression, eliciting a 150% increase at low shear stress (2 dyn/cm²) and a 70% decrease at high shear stress (12 dyn/cm²) relative to static. These differences correlated with a 60% increase in IRF-1 expression under low shear stress and a 40% decrease under high shear stress. The addition of TGRL along with cytokine activated a fourfold increase in VCAM-1 expression and a twofold increase in IRF-1 expression. The combined effect of shear stress and TGRL on the upregulation of membrane VCAM-1 was abolished by transfection of HAECs with IRF-1-specific small interfering RNA. In a healthy swine model, elevated levels of endothelial IRF-1 were also observed within atherosusceptible regions of the aorta by Western blot analysis and immunohistochemistry, implicating arterial hemodynamics in the regulation of IRF-1 expression. These data demonstrate direct roles for fluid shear stress and postprandial TGRL from human serum in the regulation of IRF-1 expression and downstream inflammatory responses in HAECs.