Atherosclerosis, the leading global cause of death, is a chronic inflammatory vascular disease with higher prevalence and earlier onset in men than in women. This study aims to investigate sex differences in the atherogenic endothelial phenotype during early atherosclerosis processes by providing the first comprehensive analysis of hormone-independent responses in human umbilical vein endothelial cells (HUVECs) from opposite-sex twins. HUVECs underwent pro-inflammatory stimulation with TNF-α and supernatant from activated pro-inflammatory THP-1 cells, revealing distinct sex-specific patterns: mRNA expression of focal adhesion proteins talin-I, vinculin, FAK, and α1-actinin increased significantly only in male cells, while paxillin showed elevated mRNA and protein levels in both sexes. Male HUVECs exhibited stronger induction of cell adhesion molecule VCAM-1, pro-inflammatory cytokine IL-1β, and proangiogenic factors Flt-3L, G-CSF, and PDGF-AA, whereas IL-22 secretion was exclusively upregulated in female cells. These sex differences in levels of focal adhesion, adhesion molecules, and cytokine profiles uncover the mechanistic backgrounds of the atherogenic endothelial phenotype, independent of systemic hormones. The findings emphasize cellular sex as a critical biological variable in early atherosclerosis and vascular inflammation.