RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.