Previous work has shown that indocyanine green (ICG)-assisted peeling of the internal limiting membrane during vitreoretinal surgery may damage the retinal pigment epithelium (RPE). The present study tested the direct toxic effects and phototoxic effects of ICG on cultured human RPE. RPE cells were exposed to ICG (0.5%, 5 min) with or without lutein (20 μM), followed by light irradiation at different doses of light energy (1.0, 3.0 and 10.0 J/cm²). After 48 h, cells were collected and stained with trypan blue to obtain the number of viable and nonviable cells in different groups. Cultures exposed to ICG without light irradiation showed a significant decrease of viable cells (−13.3%) and an increase of nonviable cells (×2.5-fold) compared with cultures not exposed to either ICG or light, indicating the presence of direct toxic effects of ICG. In cultures exposed to ICG plus light irradiation (10.0 J/cm²), viable cells decreased significantly (−45.0%) and nonviable cells increased significantly (×4.4-fold) compared with cultures exposed to ICG alone. The damage to the RPE cells depended on the dose of light (1.0–10.0 J/cm²), indicating that ICG has a phototoxic effect as well as a toxic one. Lutein, an endogenous ocular antioxidant, had a protective effect on cultures exposed to ICG and light, cells treated with leutin showed an increase of viable cells ( 74.6%) and decrease of nonviable cells (−74.4%) compared with cultures without leutin but not on cultures exposed to ICG alone. Thus, it seems that photoactivated ICG kills cells through a photoxidative mechanism. Our study suggests that preoperative oral administration of lutein may protect against the phototoxic-induced damage of ICG on the RPE cells.