Tumors are comprised of highly heterogeneous populations of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumors in vivo. These rare cancer stem cells (CSCs) have been regarded as the “bad seeds” accounted for tumor initiation, progression, metastasis, relapse and therapeutic resistance. CSC-targeted therapy seems to be a better avenue for radical cure of cancer. Deubiquitinases (DUBs), specifically disassembling ubiquitin chains, have been demonstrated to play an important role in rigidly maintaining the balance between ubiquitination and deubiquitination for protein quality control and homeostasis in normal circumstances. Dysfunction or deregulation of DUBs always leads to a series of disorders, even malignant transformation. Despite the accumulative evidence that DUB inhibitors in cancer remedy mainly target the tumor bulk, side effects like toxicity and resistance are still hard nuts to crack. In this article, we review the concept of ubiquitin proteasome system (UPS) and hallmarks of CSCs related to tumor obstinacy. We primarily summarize the CSC-related factors and signaling pathways and focus on the function of DUBs on biological traits of CSCs. We also illustrate the opportunities and challenges for the application of DUB inhibitors in the CSC-targeted therapy. Finally, we discuss the complexity of cancer stem cell hierarchy complexity and argue that a combination therapy for both CSCs and non-CSCs should be a desirable option.