Pregabalin is a lipophilic amino acid derivative of γ-amino butyric acid that displays anticonvulsant and analgesic activities against neuropathic pain. Although a role for glial cells as an important player in pain control and also as a new target for pain medicine has been suggested, the effect of pregabalin on glial cells has not been elucidated. In the present study, we have investigated the action of pregabalin on the glial cell proteome. To identify immediate early protein targets of pregabalin in glial cells, a differential proteomics approach in C6 rat glioma cells treated with pregabalin was used. Seven proteins that sensitively reacted to pregabalin treatment were identified using two-dimensional gel electrophoresis and MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS). The calcium-ion-binding chaperone, calreticulin, and the oxidative response protein, DJ-1, were up-regulated after pregabalin treatment. Hsp (heat-shock protein)-90-β, cytoskeleton protein actin and myosin also showed quantitative expression profile differences. Functionally relevant to the proteome result, immediate actin depolymerization was observed after treatment with pregabalin. These results suggest a previously undefined role of pregabalin in the regulation of chaperone activity and cytoskeleton remodelling in glial cells.