In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.