Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-α at the posttranscriptional level. To elucidate the molecular basis of TNF-α–mediated eNOS mRNA instability, eNOS 3′ untranslated region (3′-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-α–stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3′-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-α. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-α 1 (eEF1A1). In HUVECs, TNF-α rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase–eEF1A1 fusion protein specifically binds to a UC-rich sequence in the 3′-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3′-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3′-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-α–induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3′-UTR binding protein that plays a critical role in mediating TNF-α–induced decrease in eNOS mRNA stability.