In the brain, prior sublethal ischemia (preconditioning, PC) produces tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating whether and how nitric oxide (NO) produced during PC is involved in the PC-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. The rise in the extracellular concentration of glutamate during ischemia caused by the reversed uptake of glutamate (Glu) by the astrocytic Glu transporter GLT-1 was markedly suppressed by the prior PC treatment, but the suppression was reversed by treatment with an inhibitor of nitric oxide synthase (NOS) during PC. Immunocytochemical and Western blot analyses demonstrated that the expression of GLT-1 was down-regulated after the PC insult, and this down-regulation was also antagonized by treatment with NOS inhibitors during PC. Here we show that nNOS-derived NO produced during PC was crucial for the down-regulation of astrocytic GLT-1, and this down-regulation coincided with an increased survival rate of neurons.