Owing to its potent longterm neuroprotective and neurorestorative properties, glial cell line-derived neurotrophic factor (GDNF) is currently studied in neurodegenerative disease clinical trials. However, little is known about the longterm effect of GDNF on neurological recovery, brain remodeling and neuroplasticity in the post-acute phase of ischemic stroke. In a comprehensive set of experiments, we examined the effects of lentiviral GDNF administration after ischemic stroke. GDNF reduced neurological deficits, neuronal injury, blood-brain barrier permeability in the acute phase in mice. As compared with control, enhanced motor-coordination and spontaneous locomotor activity were noted in GDNF-treated mice, which were associated with increased microvascular remodeling, increased neurogenesis and reduced glial scar formation in the peri-infarct tissue. We observed reduced brain atrophy and increased plasticity of contralesional pyramidal tract axons that crossed the midline in order to innervate denervated neurons in the ipsilesional red and facial nuclei. Contralesional axonal plasticity by GDNF was associated with decreased abundance of the axonal growth inhibitors brevican and versican in contralesional and ipsilesional brain tissue, reduced abundance of the growth repulsive guidance molecule ephrin b1 in contralesional brain tissue, increased abundance of the midline growth repulsive protein Slit1 in contralesional brain tissue and reduced abundance of Slit1's receptor Robo2 in ipsilesional brain tissue. These data indicate that GDNF potently induces longterm neurological recovery, peri-infarct brain remodeling and contralesional neuroplasticity, which are associated with the fine-tuned regulation of axonal growth inhibitors and guidance molecules that facilitate the growth of contralesional corticofugal axons in the direction to the ipsilesional hemisphere. •GDNF; improves Blood-Brain Barrier permeability in acute phase of brain injury.•improves functional recovery which is associated with microvascular and tissue remodeling in subacute phase of brain injury.•increases contralesional pyramidal tract plasticity.•regulates axonal outgrowth and repulsive guidance molecules.