Ampicillin, a β-lactam antibiotic, dose-dependently protects neurons against ischemic brain injury. The present study was performed to investigate the neuroprotective mechanism of ampicillin in a mouse model of transient global forebrain ischemia. Male C57BL/6 mice were anesthetized with halothane and subjected to bilateral common carotid artery occlusion for 40 min. Before transient forebrain ischemia, ampicillin (200 mg/kg, intraperitoneally [i.p.]) or penicillin G (6,000 U/kg or 20,000 U/kg, i.p.) was administered daily for 5 days. The pretreatment with ampicillin but not with penicillin G significantly attenuated neuronal damage in the hippocampal CA1 subfield. Mechanistically, the increased activity of matrix metalloproteinases (MMPs) following forebrain ischemia was also attenuated by ampicillin treatment. In addition, the ampicillin treatment reversed increased immunoreactivities to glial fibrillary acidic protein and isolectin B4, markers of astrocytes and microglia, respectively. Furthermore, the ampicillin treatment significantly increased the level of glutamate transporter-1, and dihydrokainic acid (DHK, 10 mg/kg, i.p.), an inhibitor of glutamate transporter-1 (GLT-1), reversed the neuroprotective effect of ampicillin. Taken together, these data indicate that ampicillin provides neuroprotection against ischemia-reperfusion brain injury, possibly through inducing the GLT-1 protein and inhibiting the activity of MMP in the mouse hippocampus.