Abstract EAAT1 is a major glutamate transporter in the CNS and is required for normal neurotransmission and neuroprotection from excitotoxicity. In the present study, we have identified a novel form of the human EAAT1, named here as EAAT1ex9skip, which lacks the entire exon 9. Quantitative PCR analysis indicates that this variant is expressed throughout the CNS, both in grey matter and axonal tracts, at levels ranging between 10% and 20% of the full-length EAAT1 form. When expressed in HEK293 cells, EAAT1ex9skip mRNA is translated into a truncated protein localized in the endoplasmic reticulum. EAAT1ex9skip has no functional glutamate uptake activity but instead, exerts a dominant negative effect over full-length EAAT1 function. In turn, co-expression of full-length EAAT1 and EAAT1ex9skip variants reduces the insertion of the former into the plasma membrane. Together, these results indicate that the EAAT1ex9skip splice variant is a negative regulator of full-length EAAT1 function in the human brain.