AbstractThis work aimed to study the co‐encapsulation of ω‐3 Long‐Chain Polyunsaturated Fatty Acids (LC‐PUFAs) with β‐carotene, lutein and zeaxanthin and the cellular uptake of carotenoids. Monodisperse microcapsules with good properties, including particle surface morphology, water activity, microencapsulation efficiency, and glass transition temperature, were successfully prepared using a microfluidic‐jet spray drier with OSA‐modified starch as a wall matrix. The presence of carotenoids enhanced the oxidative stability of ω‐3 LC‐PUFAs. Caco‐2 cell uptakes of the carotenoids from the spray‐dried microcapsules were studied and compared to those encapsulated in the methylcellulose (MC) or xanthan gum (XG) delivery systems. The microcapsules encapsulated by OSA‐modified starch showed higher cellular uptakes of carotenoids (i.e., β‐carotene, 0.31 μg/mg protein; lutein, 0.52 μg/mg protein; zeaxanthin, 0.54 μg/mg protein) than those in the MC or XG delivery systems. The Caco‐2 cell uptake depended on carotenoid type, where hydrocarbon carotenoids (i.e., β‐carotene) had lower uptake than oxygenated carotenoids (i.e., lutein or zeaxanthin).