Treatment of adipose-derived stem cell (ADSC) substantially improves the neurological deficits during stroke by reducing neuronal injury, limiting proinflammatory immune responses, and promoting neuronal repair, which makes ADSC-based therapy an attractive approach for treating stroke. However, the potential risk of tumorigenicity and low survival rate of the implanted cells limit the clinical use of ADSC. Cell-free extracts from ADSC (ADSC-E) may be a feasible approach that could overcome these limitations. Here, we aim to explore the potential usage of ADSC-E in treating rat transient middle cerebral artery occlusion (tMCAO). We demonstrated that intravenous (IV) injection of ADSC-E remarkably reduces the ischemic lesion and number of apoptotic neurons as compared to other control groups. Although ADSC and ADSC-E treatment results in a similar degree of a long-term clinical beneficial outcome, the dynamics between two ADSC-based therapies are different. While the injection of ADSC leads to a relatively mild but prolonged therapeutic effect, the administration of ADSC-E results in a fast and pronounced clinical improvement which was associated with a unique change in the molecular signature suggesting that potential mechanisms underlying different therapeutic approach may be different. Together these data provide translational evidence for using protein extracts from ADSC for treating stroke.