[Purpose] In the present study, we investigated the effects of antecedent exercise on functional recovery and calpain protein expression following focal cerebral ischemia injury. [Subjects and Methods] The rat middle cerebral artery occlusion model was employed. Adult male Sprague-Dawley rats were randomly divided into 4 groups. Group I comprised untreated normal rats (n=10); Group II comprised untreated rats with focal cerebral ischemia (n=10); Group III comprised rats that performed treadmill exercise (20 m/min) training after focal cerebral ischemia (n=10); and Group IV comprised rats that performed antecedent treadmill exercise (20 m/min) training before focal cerebral ischemia (n=10). At different time points (1, 7, 14, and 21 days), limb placement test score and the levels of calpain protein in the hippocampus were examined. [Results] In the antecedent exercise group, improvements in the motor behavior index (limb placement test) were observed and hippocampal calpain protein levels were decreased. [Conclusion] These results indicated that antecedent treadmill exercise prior to focal cerebral ischemia exerted neuroprotective effects against ischemic brain injury by improving motor performance and decreasing the levels of calpain expression. Furthermore, these results suggest that antecedent treadmill exercise of an appropriate intensity is critical for post-stroke rehabilitation.