A major obstacle for the transplantation of neural stem cells (NSCs) into the lesioned spinal cord is their predominant astrocytic differentiation after transplantation. We took advantage of this predominant astrocytic differentiation of NSCs and expressed the paradigmatic beneficial neural cell adhesion molecule L1 in radial glial cells and reactive and nonreactive astrocytes as novel cellular vehicles to express L1 under the control of the promoter for the human glial fibrillary acidic protein (GFAP-L1 NSCs). Behavioral analysis and electrophysiological H-reflex recordings revealed that mice transplanted with GFAP-L1 NSCs showed enhanced locomotor recovery in comparison to mice injected with wild type (WT) NSCs or control mice injected with phosphate-buffered saline (PBS). This functional recovery was further accelerated in mice transplanted with L1-expressing radial glial cells that had been immunoisolated from GFAP-L1 NSCs (GFAP-L1-i cells). Morphological analysis revealed that mice grafted with GFAP-L1 NSCs exhibited increased neuronal differentiation and migration of transplanted cells, as well as increased soma size and cholinergic synaptic coverage of host motoneurons and increased numbers of endogenous catecholaminergic nerve fibers caudal to the lesion site. These findings show that L1-expressing astrocytes and radial glial cells isolated from GFAP-L1 NSC cultures represent a novel strategy for improving functional recovery after spinal cord injury, encouraging the use of the human GFAP promoter to target beneficial transgene expression in transplanted stem cells.