Background —Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E–deficient mice. Methods and Results —Intravenous administration of an adenovirus (5×10 ⁸ plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 ( P <0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively ( P <0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH –treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% ( P <0.001), macrophages by 69% ( P =0.006), and smooth muscle cells by 84% ( P =0.002) in the arterial wall. This resulted in a 77% reduction ( P <0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% ( P =0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice ( P =0.01 and P =0.04, respectively). Conclusions —Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E–deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.