Null mutations in the human IQCB1/NPHP5 (nephrocystin-5) gene that encodes NPHP5 are the most frequent cause of Senior-Løken syndrome, a ciliopathy that is characterized by Leber congenital amaurosis and nephronophthisis. We generated germline Nphp5-knockout mice by placing a β-Geo gene trap in intron 4, thereby truncating NPHP5 at Leu87 and removing all known functional domains. At eye opening, Nphp5−/− mice exhibited absence of scotopic and photopic electroretinogram responses, a phenotype that resembles Leber congenital amaurosis. Outer segment transmembrane protein accumulation in Nphp5−/− endoplasmic reticulum was evident as early as postnatal day (P) 6. EGFP-CETN2, a centrosome and transition zone marker, identified basal bodies in Nphp5−/− photoreceptors, but without fully developed transition zones. Ultrastructure of P6 and 10 Nphp5−/− photoreceptors revealed aberrant transition zones of reduced diameter. Nphp5−/− photoreceptor degeneration was complete at 1 mo of age but was delayed significantly in Nphp5−/−; Nrl−/−(cone only) retina. Nphp5−/− mouse embryonic fibroblast developed normal cilia, and Nphp5−/− kidney histology at 1 yr of age showed no significant pathology. Results establish that nephrocystin-5 is essential for photoreceptor outer segment formation but is dispensable for kidney and mouse embryonic fibroblast ciliary formation.—Ronquillo, CC, Hanke-Gogokhia, C., Revelo, MP, Frederick, JM, Jiang, L., Baehr, W. Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation.