Retinosomes are intracellular lipid bodies found in the retinal pigment epithelium (RPE). They contain retinyl esters (REs) and are thought to be involved in visual chromophore regeneration during dark adaptation and in case of chromophore depletion. However, key enzymes in chromophore regeneration, retinoid isomerase (RPE65), and lecithin:retinol acyltransferase (LRAT) are located in the endoplasmic reticulum (ER). The mechanism and the enzyme responsible for mobilizing REs from retinosomes remained unknown. Our study demonstrates that patatin-like phospholipase domain containing 2 (PNPLA2) mobilizes all-trans-REs from retinosomes. The absence of PNPLA2 in mouse eyes leads to a significant accumulation of lipid droplets in RPE cells, declined electroretinography (ERG) response, and delayed dark adaptation compared with those of WT control mouse. Our work suggests a function of PNPLA2 as an RE hydrolase in the RPE, mobilizing REs from lipid bodies and functioning as an essential component of the visual cycle.