To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats. Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups and B, the SCI model was established with modified Allen's method and then the rats of groups A and B were administrated by subarachnoid injection of 6 microL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord. At different time points, the BBB score of group C was significantly higher than those of groups A and B (P < 0.05), and the BBB score of group was significantly better than that of group B at 14 and 28 days after operation (P < 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P < 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P < 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P < 0.05). The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.