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To confirm the roles of D-serinergic gliotransmission in epilepsy, we investigated the relationship between spatiotemporally specific glial responses and the D-serine/serine racemase system in mesial temporal structures following status epilepticus (SE). In control animals, D-serine and serine racemase immunoreactivities were detected mainly in astrocytes. After SE, D-serine and serine racemase immunoreactivities were increased in astrocytes. Double-immunofluorescence study revealed that up-regulation of serine racemase immunoreactivity was relevant not to D-serine immunoreactivity but to nestin or vimentin immunoreactivity. Neither D-serine nor serine racemase was found in naïve or reactive microglia. In addition, phosphorylated N-methyl-D-aspartate (NMDA) receptor subunit 1 (pNR1-Ser896) immunoreactivity in the hippocampus was increased compared with controls. Increased D-serine immunoreactivity showed direct correlation with the phosphorylation of Ser896 of NR1. Given the findings of our previous study, these findings suggest that D-serine and serine racemase in astrocytes may play roles in neuronal hyperexcitability via a cooperative activation of NMDA receptors. Furthermore, serine racemase may be involved in migration and differentiation of immature astrocytes, which is relevant to reactive astrogliosis.