Background: To examine the effect of CXCR4 antagonist AMD3100 on the recruitment of endogenous endothelial precursor cells (EPC) in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) and outcome of stroke. Methods: Adult male SD rats underwent pMCAO. AMD3100 was injected once at 1 hour (an early phase) or on day 14 (a later phase) or for 7 consecutive days from day 1 to day 7(3 mg/kg/day) after pMCAO. Flow cytometry analyses were performed to detect endogenous EPCs in peripheral blood (PB). Endogenous EPCs in IBZ were identified by immunofluorescence staining. SDF-1 expression levels in IBZ were measured by real time PCR dynamically. Infarct volume and neurological outcome including neurological score and body weight loss were used to estimate the outcome of stroke. Results: AMD3100-treatment could mobilize endogenous EPCs to PB of rats after pMCAO, and continuous AMD3100-treatment mobilized more EPCs to PB than single AMD3100-treatment. Single AMD3100 treatment at 1 hour after pMCAO rather than continuous AMD3100 treatment in an early phase could recruit endogenous EPCs in IBZ and improve neurological outcome after pMCAO. Single AMD3100 administration in later phase (on day 14) could not recruit endogenous EPCs to IBZ or improve neurological outcome after pMCAO. SDF-1 relative expression in IBZ increased in an early phase from day 1 to day 3, then decreased in later phase from day 7 to day 14. Conclusions: Our findings suggested that single AMD3100 treatment in an early phase could recruit endogenous EPCs to IBZ and improve the outcome of stroke, and AMD3100 might be used for the treatment of stroke if given at proper time window.