The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis (LCA) that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In the present study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibited comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least 2-fold greater than those in age-matched rd12 mice. Moreover, FATP4-deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4-deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.Significance Statement Isorhodopsin, which employs 9-cis-retinal as the light-sensitive chromophore, is known to support rod survival and function in dark-reared Rpe65-/- mouse model of LCA that exhibits early cone degeneration. The mechanism regulating 9-cis-retinal formation and the role of 9-cis-retinal in cone survival remain largely unknown. Here, we identified FATP4 as a new negative regulator of 9-cis-retinal synthesis in RPE65-null mice. We further found that increased 9-cis-retinal synthesis did not influence rod function and degeneration, but it significantly enhanced cone survival and function in mice lacking both RPE65 and FATP4. Our findings indicate that 9-cis-retinal functions as a "cone-tropic" chromophore, providing 9-cis-retinal and FATP4 as important therapeutic targets to alleviate cone degeneration and loss of daytime color vision in RPE65 mutation-associated LCA.