The purpose of this study was to elucidate the transport mechanism(s) of L-glutamate (L-Glu), a neuroexcitatory neurotransmitter, in the inner blood–retinal barrier (BRB). The L-Glu transport was evaluated by an in vitro uptake study with a conditionally-immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells. L-Glu uptake by TR-iBRB2 exhibited time- and concentration-dependence, and was composed of high- and low-affinity processes with Michaelis–Menten constants ( K _m) of 19.3 µM and 275 µM, respectively. Under Na⁺-free conditions, L-Glu uptake by TR-iBRB2 involved one-saturable kinetics with a K _m of 190 µM, which is similar to that of the low-affinity process of L-Glu uptake under normal conditions. Moreover, substrates/inhibitors of system X_c⁻, which is involved in blood-to-retina transport of compounds across the inner BRB, strongly inhibited the L-Glu uptake under Na⁺-free conditions, suggesting that Na⁺-independent low-affinity L-Glu transport at the inner BRB is carried out by system X_c⁻. Regarding the Na⁺-dependent high affinity process of L-Glu transport at the inner BRB, L-Glu uptake by TR-iBRB2 under normal conditions was significantly inhibited by substrates/inhibitors of excitatory amino acid transporter (EAAT) 1–5, but not alanine-serine-cysteine transporters. Reverse-transcription polymerase chain reaction (RT-PCR) analysis and immunoblot analysis demonstrated that mRNA and protein of EAAT1 are expressed in TR-iBRB2 cells, whereas mRNAs and/or proteins of EAAT2-5 are not. Immunohistochemical analysis revealed that EAAT1 protein is localized on the abluminal membrane of the retinal capillaries. In conclusion, EAAT1 most likely mediates Na⁺-dependent high-affinity L-Glu transport at the inner BRB and appears to take part in L-Glu elimination from the retina across the inner BRB.