The blood-brain barrier (BBB) is a major challenge in drug delivery for the treatment of central nervous system diseases. Walnut derived peptide TWLPLPR (TW-7) has been proved to promote neuronal mitochondrial autophagy and enhance hippocampal neuronal synaptic plasticity, thereby improving learning and memory abilities in mice. We investigated the internalization mechanism and intracellular transport pathway for the walnut-derived peptide, TW-7, using bEnd.3 cells in an in vitro BBB model system. TW-7 was taken up by the bEnd.3 cells in a concentration-, temperature-, and energy-dependent manner; this involved increases in caveolin-1 and caveolin-2 protein expression and phosphorylation and inhibition of P-glycoprotein-mediated efflux. Subcellular localization of TW-7 in bEnd.3 cells was observed, indicating that the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria participated in intracellular trafficking and that the peptide escaped from lysosomes over time. Caveolae may be critical for TW-7 uptake by brain microvascular endothelial cells, assisting TW-7 to cross the BBB. The results of this study provide a theoretical basis for the mechanism of active peptide penetrating the BBB, and provide a reference for developing neuroprotective active peptide products.