Mitophagy regulates cancer stem cell (CSC) populations affecting tumorigenicity and malignancy in various cancer types. Here, we report that cisplatin treatment led to the activation of higher mitophagy through regulating CLU (clusterin) levels in oral CSCs. Moreover, both the gain-of-function and loss-of-function of CLU indicated its mitophagy-specific role in clearing damaged mitochondria. CLU also regulates mitochondrial fission by activating the Ser/Thr kinase AKT, which triggered phosphorylation of DNM1L/Drp1 at the serine 616 residue initiating mitochondrial fission. More importantly, we also demonstrated that CLU-mediated mitophagy positively regulates oral CSCs through mitophagic degradation of MSX2 (msh homeobox 2), preventing its nuclear translocation from suppressing SOX2 activity and subsequent inhibition of cancer stemness and self-renewal ability. However, CLU knockdown disturbed mitochondrial metabolism generating excessive mitochondrial superoxide, which improves the sensitivity to cisplatin in oral CSCs. Notably, our results showed that CLU-mediated cytoprotection relies on SOX2 expression. SOX2 inhibition through genetic (sh) and pharmacological (KRX-0401) strategies reverses CLU-mediated cytoprotection, sensitizing oral CSCs toward cisplatin-mediated cell death.