Background <p>Endothelial dysfunction characterized by cytokine release and adhesion molecule upregulation is a major driver of atherogenesis. Tumor necrosis factor-α (TNF-α) activates NF-κB signaling and increases ICAM-1, VCAM-1, IL-6, and IL-8 expression in endothelial cells. This study aimed to investigate whether amentoflavone (AMF) modulates TNF-α-induced inflammatory and adhesion-related responses in human endothelial cells, supporting its potential to mitigate early vascular dysfunction.</p> Methods <p>Human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α (10 ng/mL) in the presence or absence of AMF. Six experimental groups were designed to determine AMF’s prophylactic, concurrent, and post-treatment influences on inflammation. Relative mRNA levels of IL-6, IL-8, ICAM-1, VCAM-1, and NF-κB were quantified by qRT-PCR, while protein levels were measured by ELISA.</p> Results <p>TNF-α markedly increased IL-6, IL-8, ICAM-1, VCAM-1, and NF-κB expression at both the mRNA and protein levels. AMF alone did not trigger any inflammatory response and notably attenuated TNF-α-induced cytokine and adhesion molecule upregulation. Both concurrent and sequential AMF treatments reduced inflammatory responses compared with TNF-α-only cells. Prophylactic AMF administration demonstrated the greatest inhibitory effect, indicating enhanced preventive potential. AMF effectively suppresses TNF-α–mediated endothelial activation by downregulating NF-κB signaling and reducing the expression of IL-6, IL-8, ICAM-1, and VCAM-1. </p> Conclusions <p>These findings suggest that AMF may represent a promising in vitro preventive candidate against TNF-α-induced endothelial activation, warranting further validation in additional experimental models.</p> Graphical Abstract <p></p>