β-Carotene is a carotenoid with a range of reported health benefits besides vitamin A activity. If the enzymatic conversion of β-carotene to retinal is suppressed in the digestive tract, residual β-carotene that reaches the tissues increases. We evaluated the function of quercetin and rutin (quercetin-3-rutinoside) to increase the accumulation of β-carotene in vitro and in vivo in BALB/c mice. When the conversion of β-carotene by a preparation of the murine small intestine was measured in vitro, the addition of quercetin or rutin considerably inhibited the conversion. When the levels of hepatic β-carotene and retinoids were measured among three groups of mice fed a diet supplemented with β-carotene plus quercetin or rutin or β-carotene alone (four to six mice per group), quercetin increased the level of β-carotene and decreased the level of retinol, whereas rutin did not. These results demonstrate that quercetin can suppress the conversion of β-carotene which develops in the cytosol of small intestinal epithelial cells, and that rutin whose rutinose-moiety prevents being absorbed in the small intestine cannot suppress the conversion in vivo. This study offers a novel insight into the interaction between flavonoids and carotenoids with respect to the health benefits from the latter.