Mutations in the extracellular matrix protein eyes shut homolog (EYS), also known as spacemaker or spam, make up a large number of autosomal recessive cases of retinitis pigmentosa (retinitis pigmentosa 25, RP25). Drosophila EYS protein is located in and required for the formation of the inter-rhabdomeral space. Vertebrate EYS is thought to be located in the analogous extracellular space of the outer segment layer. Because the mouse genome lacks an EYS locus, we have chosen zebrafish to study the roles of EYS in photoreceptor survival. Our results showed that EYS protein was not located at the outer segment layer. EYS immunostaining exhibited a punctate pattern on the basal end of the outer segment reminiscent of connecting cilia/transition zone (CC/TZ). Double immunostaining revealed that EYS protein was located on the basal end of acetylated a-tubulin but apical to g-tubulin, indicating that EYS was localized near CC/TZ. Using CRISPR/Cas9 technology, we generated several zebrafish EYS mutant lines. Although EYS deficiency did not cause any apparent retinal defect up to 40 days post-fertilization (dpf), progressive loss of cones became apparent at 4-, 6-, and 8-months post-fertilization (mpf). Guanine nucleotide binding protein (G protein) alpha transducin activity polypeptide (GNAT) 2 in the mutant retina, as well as the numbers of red-, green-, blue-, and UV-opsin positive cones progressively decreased. TUNEL-positive nuclei were also observed in the outer nuclear layer of cone photoreceptors. Contrary to the cones, rod markers Zpr3 and GNAT1 immunostaining and GNAT1 real-time RT-PCR did not reveal any significant changes in rod photoreceptor survival. Our results indicate that EYS is located near CC/TZ and is required for cone photoreceptor survival in zebrafish.