Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced upregulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide- and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries.