Nutrients are actively taken up by the brain through various transporters at the blood–brain barrier (BBB). Orally supplied DHA must be transported from the circulating blood to the brain across the BBB through transport carriers including major facilitator superfamily domain-containing protein 2a (MFSD2A) for esterified DHA and fatty acid-binding protein 5 (FABP5) for non-esterified DHA. Although BBB integrity is known to be altered in aging, the impact of aging on DHA transport across the BBB is not well understood. Here, we used 2-, 8-, 12-, and 24-month-old male C57BL/6 mice to evaluate the brain uptake of [¹⁴C]DHA, as non-esterified form, using an in situ transcardiac perfusion technique. Primary culture of rat brain endothelial cells (RBECs) were used to evaluate the effect of siRNA-mediated MFSD2A knockdown on cellular uptake of [¹⁴C]DHA. We found that brain uptake of [¹⁴C]DHA was inhibited by an excess amount of unlabeled DHA in 2-month-old mice. Transfection of MFSD2A siRNA into RBECs decreased the protein expression levels of MFSD2A and cellular uptake of [¹⁴C]DHA. 12- and 24-month-old mice showed significant decrease in brain uptake of [¹⁴C]DHA and MFSD2A protein expression in the brain microvasculature compared with 2-month-old mice, nevertheless FABP5 protein expression was up-regulated with age. Our findings suggested that MFSD2A is involved in non-esterified DHA transport at the BBB. The middle-aged and aged brain has decreased DHA transport across the BBB due to age-related down-regulation of MFSD2A rather than FABP5.