In addition to thrombolysis, tissue plasminogen activator (tPA) is involved in synaptic plasticity, dendritic remodeling and axonal outgrowth in the developing and injured CNS. We have demonstrated that tPA administered intranasally during the subacute phase after stroke enhanced neurological recovery in rats. In the present study, we examined the therapeutic benefits of intranasal tPA treatment on corticospinal tract (CST) axonal remodeling in adult mice with transgenic yellow fluorescent protein (YFP) labeling specifically in the CST subjected to permanent middle cerebral artery occlusion (MCAo). Animals were treated intranasally with 4 doses of human recombinant tPA (300 ug/dose) or saline (control) at day 7, 9, 11 and 13 after MCAo (n=10/group), respectively. An adhesive-removal test and a single-pellet reaching test were performed at 1, 3, and 7 days after MCAo, and weekly thereafter to monitor functional recovery. Animals were euthanized 4 weeks after stroke. There was no difference in lesion volume between control and tPA-treated groups (p>0.05). Compared with saline treated animals, significant functional improvements were evident in mice treated with tPA (p<0.05), as well as significantly increased CST axonal density in the denervated side of the gray matter of the cervical cord (p<0.01). We also isolated, and cultured primary embryonic YFP neurons with tPA at concentrations of 0.065, 0.65, 2.0 and 6.5 μg/ml, respectively, for 3 days, and observed that tPA dose-dependently increased neurite length and number (P<0.05). Our data suggest that delayed tPA intranasal treatment provides therapeutic benefits for neurological recovery after stroke by, at least in part, promoting CST axonal remodeling in the denervated spinal cord gray matter.