The omega-3 fatty-acid docosahexaenoic acid (DHA) is a key component of cellular membranes in the central nervous system (CNS) and essential for neurological functions. In human cerebral cortex, DHA contributes to around 25% of total fatty acids. Deficiency of DHA results in several CNS-related disorders, such as memory deficits, dyspraxia, and dyslexia. The major source of DHA in the brain is obtained from the diet and transported in the form of lysophosphatidylcholine (LPC) across the blood-brain-barrier (BBB) by MFSD2A in a Na+-dependent manner. The detailed transport mechanism of LPC across the lipid bilayer is still elusive. Here, by using single particle cryo-EM, we revealed the structure of Danio rerio MFSD2A (59 kDa) in a complex with FAB in the inward-open conformation with ligands bound to amphipathic pockets between N- and C-terminal domains at 2.9 Å. Multiple MFSD2A structures with individual ligands located at unique positions along the translocation pathway were further identified by extensive classification of particles. These structures depicted the DHA-LPC translocating trajectory along the cavity, where a ∼70 degree rotation of LPC headgroup followed by translation of the ligand towards the cytoplasmic exit occurred after the initial lipid flipping step. Together these results provide a detailed model for omega-3 fatty-acid transport and potential for the design of the delivery strategies for amphipathic drugs across the BBB.