SignificancePreclinical evaluation of retinal pigment epithelial (RPE) cell–based therapy has largely been dependent on qualitative in vivo ophthalmic imaging techniques and ex vivo histological characterization of transplanted cells. However, these readouts do not provide a comprehensive picture of gene expression changes at single-cell resolution. In this study, we transplanted RPE monolayers in the subretinal space of immunocompetent rabbits and performed single-cell RNA sequencing. We observed unequivocal maturation toward the native adult human RPE state, and identified key regulons activated upon transplantation. These key regulons may confer RPE canonical functions important for retinal homeostasis and promote cell survival in vivo. These findings shed insights into the behavior of RPE after subretinal transplantation, with important implications for cell-based therapy for age-related macular degeneration.