In this study, we evaluated whether propentofylline, a methylxanthine derivative, modulates spinal glial activation and GABAergic inhibitory tone by modulation of glutamic acid decarboxylase (GAD) 65, the GABA synthase enzyme, in the spinal dorsal horn following spinal cord injury (SCI). Sprague–Dawley rats (225–250 g) were given a unilateral spinal transverse injury, from dorsal to ventral, at the T13 spinal segment. Unilateral spinal injured rats developed robust bilateral hindlimb mechanical allodynia and hyperexcitability of spinal wide dynamic range (WDR) neurons in the lumbar enlargement (L4–L5) compared to sham controls, which was attenuated by intrathecal (it) administration of GABA, dose-dependently (0.01, 0.1, 0.5 μg). Western blotting and immunohistochemical data demonstrated that the expression level of GAD 65 protein significantly decreased on both sides of the lumbar dorsal horn (L4/5) after SCI (p< 0.05). In addition, astrocytes and microglia showed soma hypertrophy as determined by increased soma area and increased GFAP and CD11b on both sides of the lumbar dorsal horn compared to sham controls, respectively (p< 0.05). Intrathecal treatment with propentofylline (PPF 10 mM) significantly attenuated the astrocytic and microglial soma hypertrophy and mechanical allodynia (p< 0.05). Additionally, the Western blotting and immunohistochemistry data demonstrated that it treatment of PPF significantly prevented the decrease of GAD 65 expression in both sides of the lumbar dorsal horn following SCI (p< 0.05). In conclusion, our present data demonstrate that propentofylline modulates glia activation and GABAergic inhibitory tone by modulation of GAD 65 protein expression following spinal cord injury.