Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin-1 (PS1) as a regulator of glutamate transport. PS1-deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild-type neurons. Gamma-secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss-of-function, accomplished by PS1 deficiency or γ-secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF-96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss-of-function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage-gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.