Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival, synaptic plasticity, learning and memory, and neuroplasticity. Further, exogenous treatment with BDNF or exposing animals to enrichment and exercise regimens, which also increase BDNF, enhances behavioral recovery after brain injury. Thus, the beneficial effects of rehabilitation in promoting recovery after stroke may also depend on BDNF. We tested this hypothesis by evaluating the contribution of BDNF to motor skill relearning after endothelin-1-induced middle cerebral artery occlusion in rats. Antisense BDNF oligonucleotide, which blocks the expression of BDNF (or saline vehicle) was infused into the contralateral lateral ventricle for 28 days after ischemia. Animals received either a graduated rehabilitation program, including running exercise and skilled reaching training, which simulates clinical practice, or no rehabilitation. Functional recovery was assessed with a battery of tests that measured skilled reaching, forelimb use asymmetry, and foraging ability. Rehabilitation significantly improved skilled reaching ability in the staircase task. Antisense BDNF oligonucleotide effectively blocked BDNF mRNA, and negated the beneficial effects of rehabilitation on recovery of skilled reaching. Importantly, antisense BDNF oligonucleotide did not affect reaching with the unaffected limb, body weight, infarct size, or foraging ability, indicating the treatment was specific to relearning of motor skill after ischemia. This study is the first to identify a critical role for BDNF in rehabilitation-induced recovery after stroke, and our results suggest that new treatments to enhance BDNF would constitute a promising therapy for promoting recovery of function after stroke.