A Type II pyrethroid pesticide β-cypermethrin is widely used in agriculture and domestic applications for pest control. However, the effect of β-cypermethrin on the glutamate neurotransmitter has not been well-documented. In the current study, mice were treated with 20, 40, or 80 mg/kg β-cypermethrin by a single oral gavage, with corn oil as a vehicle control. Four hours after treatment, we investigated glutamate levels and glutamate-metabolizing enzyme (phosphate-activated glutaminase, PAG; glutamine synthetase, GS) activities in the cerebral cortex of mice, using a HPLC system with ultraviolet detectors and a colorimetric assay. Glutamate uptake levels in the synaptosomes of cerebral cortex and mRNA expression levels of PAG, GS, and glutamate transporter-1 (GLT-1) in the cerebral cortex were detected by a radioactive labeling method and qRT-PCR, respectively. Toxic symptoms were observed in mice treated with 40 or 80 mg/kg β-cypermethrin. Compared with the control, significant decreases in glutamate level and GS activity, and an obvious increase in synaptosomal glutamate uptake, were found in the cerebral cortex of mice treated with 80 mg/kg β-cypermethrin. No significant changes were found among groups in PAG activity or PAG, GS, and GLT-1 mRNA expression levels. These results suggest that β-cypermethrin treatment may reduce the glutamate level in the mouse cerebral cortex, which is associated with decreased GS activity and increased synaptosomal glutamate uptake. Our findings provide a partial explanation for the neurotoxic effects of synthetic β-cypermethrin insecticides.