Synthetic 9-cis-stereoisomers of vitamin A (all-trans-retinol) are especially promising agents for the fight against blinding diseases. Several studies suggested that 9-cis-β,β-carotene (9-cis-BC), a natural and abundant β-carotene isomer in the diet, could be the precursor of 9-cis-retinoids and thus could have therapeutic applications. Here we showed that 9-cis-BC is metabolized both in vitro and in vivo by two types of mouse carotenoid oxygenases, β,β-Carotene monooxygenase 1 (BCMO1), and β,β-carotene dioxygenase 2 (BCDO2). In the symmetric oxidative cleavage reaction at C15,C15' position by BCMO1, part of the 9-cis-double bond was isomerized to the all-trans-stereoisomer, yielding all-trans-retinal and 9-cis-retinal in a molar ratio of 3:1. The asymmetric cleaving enzyme BCDO2 preferentially removed the 9-cis-ring site at the C9,C10 double bond from this substrate, providing an all-trans-β-10'-apocarotenal product that can be further metabolized to all-trans-retinal by BCMO1. Studies in knockout mouse models confirmed that each carotenoid oxygenase can metabolize 9-cis-BC. Therefore, treatment of mouse models of Leber congenital amaurosis with 9-cis-BC and 9-cis-retinyl-acetate, a well established 9-cis-retinal precursor, showed that the cis-carotenoid was far less effective than the cis-retinoid in rescuing vision. Thus, our in vitro and in vivo studies revealed that 9-cis-BC is not a major source for mouse 9-cis-retinoid production but is mainly converted to all-trans-retinoids to support canonical vitamin A action.