Spinal cord injury (SCI) initiates a cascade of processes that ultimately form a nonpermissive environment for axonal regeneration. Emerging evidence suggests that regenerative failure may be due in part to inhibitory factors expressed by reactive spinal cord glial cells and meningeal fibroblasts, such as the Eph receptor protein-tyrosine kinases and their corresponding ligands (ephrins). Here we sought to assess the role of ephrin B2, an inhibitory axonal guidance molecule, as an inhibitor of the recovery process following SCI. To determine the extent of ephrin B2 involvement in axonal regenerative failure, a SCI model was performed on a conditional ephrin B2 knockout mouse strain (ephrin B2(-/-)), in which the ephrin B2 gene was deleted under the GFAP promoter. The expression of ephrin B2 was significantly decreased in astrocytes of injured and uninjured ephrin B2(-1-) mice compared to wild-type mice. Notably, in the ephrin B2(-/-) mice, the deletion of ephrin B2 reduced astrogliosis, and accelerated motor function recovery after SCI. Anterograde axonal tracing on a hemisection model of SCI further showed that ephrin B2(-/-) mice exhibited increased regeneration of injured corticospinal axons and a reduced glial scar, when compared to littermate controls exposed to similar injury. These results were confirmed by an in vitro neurite outgrowth assay and ephrin B2 functional blockage, which showed that ephrin B2 expressed on astrocytes inhibited axonal growth. Combined these findings suggest that ephrin B2 ligands expressed by reactive astrocytes impede the recovery process following SCI. (c) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.