Technical Abstract: Epidemiological studies have shown that consumption of carotenoid-rich fruits and vegetables is associated with reduced risk of developing chronic diseases. B-carotene, a-carotene and b-cryptoxanthin are precursors of vitamin A, a nutrient essential for human health. However, little is known about the bioavailability of carotenoids from whole foods. This study characterized the intestinal uptake performance of carotenoids using monolayers of differentiated Caco-2 human intestinal cancer cells and mimicked human digestion to assess carotenoid absorption from carrots and corn. Results showed that Caco-2 cellular uptake of b-carotene and zeaxanthin was higher than that of lutein. Uptake performance of pure carotenoids and carotenoids from whole foods by Caco-2 cells was both curvilinear, reaching saturated levels after 4 hours of incubation. The time kinetics and dose response of carotenoid uptake presented a similar pattern in Caco-2 cells after plating for 2 days and 14 days. Furthermore, we verified the applicability of this new model with whole grain-corns, showing that cooked corn grain significantly enhanced carotenoid bioavailability. These results support the feasibility of the in vitro digestion cell model for assessing carotenoid absorption from whole foods as a suitable and cost-effective physiological alternative to current methodologies.