Annatto is a natural pigment widely used in the food industry to add yellow to red colors to dairy and cereal products. Here, the in vitro bioaccessibility and potential bioavailability of norbixin, the abundant 24-carbon carotenoid in annatto, were investigated. Norbixin added to milk was highly stable during simulated digestion, and bile salts enhanced partitioning of this carotenoid in the aqueous fraction during the small intestinal phase of digestion. Apical uptake of norbixin by Caco-2 cells was proportional to the concentration in apical medium, but cellular content increased only slightly after 60 min. Transport of norbixin to the basolateral compartment was maximum at 120 min. Both all-trans and cis isomers of norbixin were present in cells and basolateral medium. The results suggest that ingested norbixin is stable during gastric and small intestinal phases of digestion and that both cis and all-trans isomers are bioavailable.