Cathepsin S is a lysosomal cysteine protease that is overexpressed in various cancer models and plays important role in tumorigenesis, however the mechanisms are unclear. In the present study, we found that inhibition of cathepsin S induced autophagy and mitochondrial apoptosis in human glioblastoma cells. Blockade of autophagy by either a chemical inhibitor or RNA interference attenuated cathespin S inhibition-induced apoptosis. Furthermore, autophagy and apoptosis induction was dependent on the suppression of phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) signaling pathway and activation of c-Jun N-terminal kinase (JNK) signaling pathway. In addition, reactive oxygen species (ROS) served as an upstream of PI3K/AKT/mTOR/p70S6K and JNK signaling pathways. In conclusion, the current study revealed that cathepsin S played an important role in the regulation of autophagy and apoptosis in human glioblastoma cells.