Cancer stem cells (CSCs) as a small subpopulation in tumor bulk are believed to initiate tumor formation and are responsible for the resistance to cancer therapy. The proliferation and differentiation of CSCs result in heterogeneity in a tumor which increases the chance of tumor survival and invasion. Many signaling pathways are abnormally activated or repressed in CSCs. Understanding these pathways and the metabolisms in CSCs may help targeted therapy in drug-resistant tumors. The PI3K/Akt/mTOR pathway is one of the major signaling pathways in CSCs involved in the maintenance of stemness, proliferation, differentiation, epithelial to mesenchymal transition (EMT), migration, and autophagy. Thus, suppressing the PI3K/Akt/mTOR pathway with inhibitors might be a promising strategy for targeted cancer therapy. Although the pathway is well-recognized and reviewed in tumor bulks, the functions in CSCs have not been well focused. Here, we reviewed the PI3K/Akt/mTOR signaling pathway and its functions in CSCs and addressed the potential therapeutic applications in drug-resistant tumors.