The roles of cathepsins in the ischemic astrocytic injury remain unclear.Here we test the hypothesis that the activation of cathepsin B and cathepsin L, two major cathepsins in neurons, contribute to astrocytic injury via inhibiting cathepsins-mitochondrial apoptotic signaling pathways during acute focal ischemia and oxygenglucose deprivation (OGD).A stroke or a paradigm of ischemic insult was induced in adult Sprague Dawley rats by permanent middle cerebral artery occlusion (pMCAO), or in the primary cultured astrocytes by OGD, respectively.In the rat models of pMCAO, CA-074Me, a selective inhibitor of cathepsin B, or Clik148, a selective inhibitor of cathepsin L, administrated before ischemia or at 6 h after ischemia, reduced the infarct volume, improved the neurological deficits and increased the expression of MAP2 and GFAP at 24 h after ischemia.In OGD-induced primary cultured astrocytes injury, CA-074Me or Clik148 significantly decreased the LDH leakage and increased the number of astrocytes in a dose-dependent manner, and also increased the expression of GFAP.CA-074Me or Clik148 has no markedly effects on non-OGD astrocytes.In ischemic cortex, pMCAO induced an increase in activated cathepsin B or cathepsin L, tBid and caspase-3, reduced Cyt-c in mitochondria and increased Cyt-c in cytoplasm.CA-074Me or Clik148 reversed pMCAO-induced increases in activated cathepsin B or cathepsin L, tBid and Caspase-3, and a reduction in mitochondrial Cyt-c and an increase in cytoplastic Cyt-c.Immunofluorescence showed that the dots distribution of cathepsin L or cathepsin B was seen in astrocytes in the ischemic cortex of sham, while the diffussion distribution of cathepsin L or B was seen in astrocytes in the ischemic cortex of ischemic control rats at 3 h or 6 h after ischemia, suggesting the release of cathepsin L or cathepsin B from lysosome to cytosol after ischemia and the activation of cathepsin L or cathepsin B.pMCAO also induced the activation of caspase-3 in ischemic astrocytes at 12 h after ischemia.CA-074Me or Clik148 reversed pMCAO-induced activation of cathepsin B or cathepsin L and caspase-3 in ischemic astrocytes.Western Blot analysis showed that OGD induced an increase in activated cathepsin B or cathepsin L, tBid and Caspase-3, reduced Cyt-c in mitochondria and increased Cyt-c in cytoplasm in astrocytes.CA-074Me or Clik148 blocked OGD-induced an increase in activated cathepsin B or cathepsin L, tBid and caspase-3, and a reduction in mitochondria Cyt-c and an increase in cytoplasm Cyt-c.These results suggest that targeting prodeath signaling upstream of mitochondria damage-cathepsins in ischemic astrocytes contributes to neuroprotection against cerebral ischmia via blocking the activation of cathepsins-mitochondrial apoptotic signaling pathway.