Glutamate is an essential neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of major glutamate transporters primarily expressed in astroglial cells. Dysfunction of EAAT2 is implicated in acute and chronic neurological disorders, including stroke/ischemia, temporal lobe epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, HIV-1-associated dementia, and growth of malignant gliomas. We previously discovered that ceftriaxone, one of the ß-lactam antibiotics, is a stimulator of EAAT2 expression with neuroprotective effects both in in vitro and in vivo models based in part on its ability to inhibit neuronal cell death by glutamate excitotoxicity. Based on this consideration and its lack of toxicity, ceftriaxone has potential to manipulate glutamate transmission and ameliorate neurotoxicity. In the present study, we investigated the mechanism by which ceftriaxone enhances EAAT2 expression in primary human fetal astrocytes (PHFA). Ceftriaxone significantly elevated EAAT2 transcription in PHFA through the nuclear factor-B (NF-B) signaling pathway. The antibiotic promoted nuclear translocation of p65 and activation of NF-B. The specific NF-B binding site at –272 position of the EAAT2 promoter was responsible for ceftriaxone-mediated EAAT2 induction. In addition, ceftriaxone increased glutamate uptake, a primary function of EAAT2, and EAAT2 siRNA completely inhibited ceftriaxone-induced glutamate uptake activity in PHFA. Taken together our data indicate that ceftriaxone is a potent modulator of glutamate transport in PHFA through NF-B-mediated EAAT2 promoter activation. These findings suggest a mechanism for ceftriaxone modulation of glutamate transport and for its potential effects on ameliorating specific neurodegenerative diseases, such as amyotrophic lateral sclerosis, through modulation of extracellular glutamate levels.