In addition to thrombolysis, tissue plasminogen activator (tPA) can evoke neurorestorative processes. We therefore investigated the therapeutic effect of subacute intranasal administration of tPA post stroke on neurological recovery and on corticospinal innervation in mice. A transgenic mouse line, in which the pyramidal neurons and corticospinal tract (CST) axons are specifically labeled by yellow fluorescent protein (YFP) was employed. Adult CST-YFP mice were subjected to right unilateral middle cerebral artery occlusion (MCAo), and were randomly divided into groups treated with saline or tPA intranasally in the subacute phase. Pseudorabies virus (PRV)-614-monomeric red fluorescent protein (RFP) was injected into the left forelimb. The cervical spinal cord and brain were processed for fluorescent microscopy to detect YFP and RFP labeling. Primary embryonic neurons were cultured with tPA at different concentrations. Neurite length and branch numbers were then measured. In vivo, subacute tPA treatment significantly enhanced functional recovery (p<0.05), and increased CST density in the denervated gray matter, and in the numbers of PRV-labeled neurons in bilateral cortices. The behavioral performance was significantly correlated with axonal density in the denervated spinal cord. In vitro, both neurite length and branch numbers significantly increased with concentration of tPA (p<0.05). Our results demonstrate that tPA dose-dependently increases neurite outgrowth and branching of cultured cortical neurons. Subacute intranasal administration of tPA may provide enhance neurological recovery after stroke by promoting CST axonal remodeling.