Recombinant tissue plasminogen activator (tPA) is a Food and Drug Administration-approved thrombolytic treatment for ischemic stroke. tPA is also naturally expressed in glial and neuronal cells of the brain, where it promotes axon outgrowth and synaptic plasticity. However, there are conflicting reports of harmful versus neuroprotective effects of tPA in acute brain injury models. Furthermore, its impact on white matter integrity in preclinical traumatic brain injury (TBI) has not been thoroughly explored, although white matter disruption is a better predictor of long-term clinical outcomes than focal lesion volumes. Here we show that the absence of endogenous tPA in knockout mice impedes long-term recovery of white matter and neurological function after TBI. tPA-knockout mice exhibited greater asymmetries in forepaw use, poorer sensorimotor balance and coordination, and inferior spatial learning and memory up to 35 d after TBI. White matter damage was also more prominent in tPA knockouts, as shown by diffusion tensor imaging, histological criteria, and electrophysiological assessments of axon conduction properties. Replenishment of tPA through intranasal application of the recombinant protein in tPA-knockout mice enhanced neurological function, the structural and functional integrity of white matter, and postinjury compensatory sprouting in corticofugal projections. tPA also promoted neurite outgrowth in vitro, partly through the epidermal growth factor receptor. Both endogenous and exogenous tPA protected against white matter injury after TBI without increasing intracerebral hemorrhage volumes. These results unveil a previously unappreciated role for tPA in the protection and/or repair of white matter and long-term functional recovery after TBI.