An HLF (HIF-1α-like factor)/HIF-2α-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF kd/kd) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF kd/kd mice expressing 80–20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia–normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF kd/kd mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF kd/kd mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF kd/kd mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF kd/kd mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.