The increased accumulation of amyloid b (Ab) in retinal blood vessels in Alzheimer’s disease (AD) patients and AD animal models is in significant correlation with the brain amyloid deposits associated with the cognitive impairment in AD. Ab accumulation in blood vessels can be the result of impaired transcytosis and/or the dysfunctional ocular glymphatic system in AD. The supplementation with high-dose fish oil (FO) have shown significant effects in diminishing AD pathologies but also serving as the blood vessel fortifier. We analyzed the changes in expression of major facilitator superfamily domain-containing protein2a (Mfsd2a), the main regulator of transcytosis, and of Aquaporin4 (Aqp4), the key player implicated in the functioning of the glymphatic system, in the retinas of 5xFAD female mice supplemented with the high dose fish oil for three weeks. A strong increase in Mfsd2a expression was observed in 4 M 5xFAD retinas supplemented with FO in comparison to the control 5xFAD mice. The decrease in the expression of srebp1-c could be at least partially responsible for the Mfsd2a increased expression. The observed increase in Aqp4 expression in 4M 5xFAD retinas indicative of the impaired glymphatic system was abolished with the FO supplementation. Consequently, the accumulation of Ab in 5xFAD retinal blood vessels was diminished following the FO supplementation. These findings indicate that the high-dose FO supplementation can serve as an adjuvant in developing new treatments that can improve the regulation of transcytosis or the function of glymphatic system in AD retina.