BACKGROUND: Retinopathy of prematurity (ROP) is the major cause of blindness in children. It is a biphasic disease with retinal vessel growth cessation and loss (Phase I) followed by uncontrolled retinal vessel growth (Phase II). Folate is an essential nutrient for fetal development and growth. Premature infants have a high risk for folate deficiency. However, the contribution of folate to ROP risk remains unknown. METHODS: In mouse oxygen-induced retinopathy (OIR), the nursing dams were fed with a folic acid-deficient or control diet after delivery until the end of hyperoxia. Alternatively, pups received direct injection of either folic acid or vehicle during Phase I hyperoxia. Genes involved in the folate cycle and angiogenic responses were examined using real-time PCR. Total retinal folate levels were measured with the RESULTS: Maternal folic acid deficiency in early life exacerbated pathological retinal vessel growth, while supplementation with folic acid suppressed it. Genes involved in the folate cycle were downregulated in Phase I OIR retinas and were highly expressed in Müller glia. Folic acid reduced pro-angiogenic signaling in cultured rat retinal Müller glia in vitro. CONCLUSIONS: Appropriate supplementation of folic acid might be a new and safe treatment for ROP at an early stage.