Ginkgolide B (GB) has been shown to exert neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury. However, the underlying mechanism by which GB prevents ischemic cell death remains unclear. Lysosomal proteases, including cathepsins B and L, have been implicated in ischemic cell death following reperfusion. Therefore, in the present study, we investigated the role of GB with respect to cathepsin-mediated cell death following I/R. Both the expression and enzymatic activity of cathepsins B and L were significantly increased in the ischemic cortex following cerebral I/R injury. We found that GB treatment markedly decreased the activity and expression of cathepsins B and L following I/R. Moreover, GB reduced necrotic and apoptotic cell death following I/R. These data strongly suggest that GB prevents cathepsin-mediated cell death following focal cerebral I/R injury, and they might provide new insights into the mechanism of the neuroprotective effects of GB.