The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light-emitting diode (LED) modules with peaks at either 411 nm (half bandwidth 17 nm) or 470 nm (half bandwidth 25 nm) at defined irradiances of 0.6, 1.5 and 4.5 W/m2 for 411 nm and 4.5 W/m2 for 470 nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411 nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411 nm light at 4.5 W/m2. In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411 nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase-3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above-mentioned parameters were not observed when cells were irradiated with 471 nm light despite a high irradiance of 4.5 W/m2, indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411 nm (4.5 W/m2) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411 nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.