ABSTRACTAs a member of thefibronectin leucine-rich transmembrane(flrt) gene family,fibronectin leucine-rich transmembrane 2(flrt2) is strongly expressed in a subset of sclerotome cells, and the resultant protein interacts with FGFR1 in the FGF signaling pathway during development. Studies onflrt2have focused mainly on its roles in the brain, heart and chondrogenesis. However, reports on its expression and function in the zebrafish retina are lacking. Here, we detected the high expression offlrt2in zebrafish retina usingin situhybridization technique and developed anflrt2-knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. Quantitative real-time PCR was used to measure the expression levels offlrt2, which results in an approximately 60% mRNA reduction. Theflrt2-KO zebrafish eyes’ altered morphological, cellular, and molecular events were identified using BrdU labeling, TUNEL assay, immunofluorescent staining, fluorescent dye injection and RNA sequencing. Abnormal eye development, known as microphthalmia, was found inflrt2-KO larvae, and the retinal progenitor cells exhibited increased apoptosis, perhaps owing to the combined effects ofcrx,neurod4,atoh7, andpcdh8downregulation andCasp3aandCaspblupregulation. In contrast, the retinal neural development, as well as retinal progenitor cell differentiation and proliferation, were not affected by theflrt2deletion. Thus,flrt2appears to play important roles in retinal development and function, which may provide the basis for further investigations into the molecular mechanisms of retinal development and evolution.