Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. There are two forms of AMD, dry and wet, the latter so named because of the presence of choroidal neovascularisation. Both forms lead to retinal pigment epithelial cell (RPE) atrophy and photoreceptor cells loss. AMD is characterised by the presence of drusen, deposits mainly of lipids and cholesterol. At the genetic level AMD is associated with several single nucleotide polymorphisms in gene encoding proteins involved in lipid biosynthesis, including ATP-binding cassette transporter A1 (ABCA1). Likewise, metabolic studies support the role of lipids, including cholesterol in the disease process. RPE cells process large amounts of lipids mainly derived from photoreceptor cells. In this paper, cholesterol efflux was measured in RPE cells, in human and mouse eyecups and the ARPE-19 cell line. The results show that ABCA1 was present on both sides of polarised RPE cells. This was functional as shown by directional efflux of labelled cholesterol deliver to cells. Inhibition of ABCA1 via neutralising antibody or boosting via a liver-X receptor agonist confirmed the role of ABCA1 by decreasing or increasing cholesterol transport respectively. These results suggest that ABCA1-mediated cholesterol efflux is a critical process in lipid homeostasis in the retina, and that alterations in this pathway are relevant to AMD development or progression.