Background: Inflammatory responses are important mechanisms that are involved in cerebral ischemia/reperfusion(I/R) injury. Whether toll-like receptor 9(TLR9), which belongs to the innate immune system, takes part in the inflammatory responses following cerebral I/R remains unclear.Method: This study examined the effect of different dosages of the TLR9 antagonist inhibitory oligodeoxynucleotide (iCpG-ODN) on cerebral I/R injury by using a mouse model of transient middle cerebral artery occlusion. Neurological function, infarct size, splenocytes and the expression of TLR9 and the downstream products of the TLR9 pathways were determined after cerebral I/R for up to 72 hours. Results: The Clark's focal symptom scoring showed iCpG-ODN improved neurological deficits following focal cerebral I/R. The iCpG-ODN administration significantly decreased the infarct size in a dose-dependent manner. RT-PCR showed that iCpG-ODN attenuated the I/R-induced RNA expression of TLR9. Immunoblot showed that iCpG-ODN prevented I/R-induced increases in NFκB and IRF7 levels and that it further downregulated the levels of IL-1β, TNF-α, and INF-β in the brain. iCpG-ODN did not alter the levels of TNF-α or INF-β in the peripheral blood or affect stroke-induced changes in the number of splenocytes. Conclusion: These findings suggest that iCpG-ODN induced protection against cerebral I/R via inhibiting inflammatory responses in a dose-dependent manner and may be useful in therapy for stroke patients.