High-density lipoproteins and their major protein constituent apolipoprotein A-I (apoA-I) possess diverse atheroprotective properties. Most of them must be exerted within the arterial wall. Actually, high-density lipoproteins are the most abundant lipoproteins within the arterial intima. We have recently reported that apoA-I is transcytosed through aortic endothelial cells. In the present study, we evaluate the role of ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor BI (SR-BI) in this process. Using pharmacological interventions and RNA interference, we investigated whether ABCA1 and SR-BI modulate apoA-I binding, internalization and transcytosis in endothelial cells. Upregulation of ABCA1 with oxysterols increased apoA-I binding and internalization. Trapping ABCA1 on the cell surface with cyclosporin A enhanced apoA-I binding but decreased its internalization and transcytosis. In addition, apoA-I binding, internalization, and transcytosis were reduced by at least 50% after silencing ABCA1 but not after knocking down SR-BI. The integrity of the endothelial cell monolayer was affected neither by cyclosporin A treatment nor by ABCA1 silencing, as controlled by measuring inulin permeability. Finally, in ABCA1-GFP-expressing cells, fluorescently labeled apoA-I colocalized intracellularly with ABCA1-GFP. However, apoA-I-containing vesicles did not colocalize with the late endosome marker LAMP-1 (lysosome-associated membrane protein-1). In conclusion, ABCA1, but not SR-BI, modulates the transcytosis of apoA-I through endothelial cells.