Cholesterol is the most abundant lipid component of the plasma membrane, and thus the equilibrium between free cholesterol and raft cholesterol act as a determinant of raft function and cell signalling. The mechanisms that regulate the lipid raft cholesterol levels are largely unknown. Here we demonstrate that SERPINA1 (alpha1-antitrypsin), an acute phase protein and the classical neutrophil elastase inhibitor, is localized within lipid rafts in primary human monocytes in vitro. SERPINA1 association with monocytes is inhibited by cholesterol depleting/efflux-stimulating agents (nystatin, filipin, MbetaCD (methyl-beta-cyclodextrin) and oxidized low-density lipoprotein (oxLDL) and conversely, enhanced by free cholesterol. Furthermore, SERPINA1/monocyte association per se depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, formation of cytosolic lipid droplets, and a complete inhibition of oxLDL uptake by monocytes. Our findings for the first time highlight that the entry and cell association of SERPINA1 is dependent on lipid raft cholesterol and that SERPINA1 depletes lipid raft cholesterol.