This paper reviews the preclinical effects of brilliant blue G (BBG) on the morphology and functions of the retina, and reports on a pilot study of BBG staining and subsequent peeling of the internal limiting membrane (ILM) during vitreoretinal surgery. BBG solution was injected into rat eyes and investigated using light microscopy and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, and electroretinography (ERG). No pathological changes were caused by the BBG intravitreous injection. Although electron microscopy revealed that high doses of BBG induced vacuolization in the inner retinal cells, there was no reduction in the amplitude of the ERG waves and no detectable toxic effects. In the primate eyes, the ILM was clearly visualized by BBG staining, and peeled off easily from the retina. In the clinical study, BBG improved visualization of the ILM, allowing peeling and surgery to be performed successfully on patients with various vitroretinal diseases. Improvement of postoperative visual acuity was obtained in most cases, and no adverse effects were observed postoperatively. In conclusion, BBG has low toxicity, high staining ability, and is easy to handle, making it a good candidate dye for visualizing vitreoretinal disease surgery without adverse effects.