Heme oxygenase-1 (HO-1; encoded by the Hmox1 gene) catalyzes the degradation of free heme into biliverdin, via a reaction that releases iron (Fe) and carbon monoxide. We report that HO-1 down-regulates the proinflammatory phenotype associated with endothelial cell (EC) activation by reducing intracellular nonprotein-bound Fe (labile Fe). EC isolated from Hmox1−/− mice have higher levels of intracellular labile Fe and reactive oxygen species (ROS) as compared with EC isolated from Hmox1+/+ mice. Basal and TNF-induced expression of VCAM-1, ICAM-1, and E-selectin were increased in Hmox1−/− vs Hmox1+/+ EC, an effect reversed by Fe chelation using deferoxamine mesylate (DFO). Fe chelation inhibits TNF-driven transcription of Vcam-1, Icam-1, and E-selectin, as assessed using luciferase reporter assays. This effect is associated with inhibition of the transcription factor NF-κB via a mechanism that is not associated with the inhibition of IκBα phosphorylation/degradation or NF-κB (ie, RelA) nuclear translocation, although it affects very modestly NF-κB binding to DNA κB consensus sequences in the Vcam-1 and E-selectin promoters. HO-1 inhibits NF-κB (ie, RelA) phosphorylation at Ser 276, a phosphoacceptor that is critical to sustain TNF-driven NF-κB activity in EC. This effect was mimicked by Fe chelation as well as by antioxidants (N-acetylcysteine). In conclusion, we demonstrate a novel mechanism via which HO-1 down-modulates the proinflammatory phenotype of activated EC, ie, the inhibition of RelA phosphorylation at Ser 276.