Glutamine synthetase (GS) is an enzyme involved in an endogenous mechanism of protection against glutamate neurotoxicity and is important in the regulation of astrocyte migration. To date, limited information is available concerning the expression of GS in normal spinal cords and following injury. In the present study, GS expression was identified in astrocytes, oligodendrocytes and microglia in normal rat spinal cords. Following traumatic spinal cord injury (SCI), the glutamate concentration increased rapidly at 1 h and returned to baseline rapidly. However, the GS activity and protein levels were found to decrease at 4 h and then increase gradually from day 3 following SCI. The quantification of astrocytes, oligodendrocytes and activated microglia/macrophages, as well as immunohistochemistry staining of day 7 post‑injured spinal cords, indicated that the astrocytes and microglia/macrophages contributed to the increase in GS. Collectively, the results provided evidence for the temporospatial expression and location of GS following SCI and suggested that the changes in GS levels may contribute to glutamate neurotoxicity and glial cell response following SCI.