This study aims to explore the effect of picroside Ⅱ on the expression of matrix metalloproteinase-9 (MMP-9) and optimize the therapeutic dose and time window after cerebral ischemic injury in rats.The forebrain ischemia models of Wistar rats were randomly grouped according to orthogonal experimental design of [L16(45)] and treated by injecting picroside Ⅱ intraperitoneally with different doses at various times.The morphology and ultrastructure of brain tissue were observed by haematoxylin eosin (HE) staining.The expression of MMP-9 was determined by immunohistochemical (IHC) staining and Western blotting (WB).The expression of MMP-9 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).The Results indicated that the expression of MMP-9 was reduced obviously and the structure of brain tissue was improved obviously than those in model group after treated by picroside Ⅱ.The optimal therapeutic dose and time window of picroside Ⅱ in treating cerebral ischemic injury were ischemia 2.0h with 10mg/kg body weight according to the results of HE and IHC staining, ischemia 1.5h with 10mg/kg according to WB and ischemia 1.5h with 20mg/kg according to RT-PCR results respectively.It is concluded that picroside Ⅱ could protect the blood brain barrier and brain tissue against ischemic injury by reducing the expression of MMP-9 after cerebral ischemic injury.Based on the orthogonal experimental results, the best therapeutic dose and time window of picroside Ⅱ should be injected intraperitoneally with 10-20mg/kg body weight at cerebral ischemia 1.5-2.0h.