Tumor-initiating stem cells (TiSCs) are key drivers of tumor resistance, relapse, and immune evasion, with mitochondria playing a central role in regulating these processes. TiSCs and cancer stem cells (CSCs) rely heavily on oxidative phosphorylation (OXPHOS) for ATP production while maintaining metabolic plasticity, enabling them to switch between glycolysis and OXPHOS depending on nutrient availability and oxygen levels in the tumor microenvironment (TME). This adaptability enhances their ability to survive harsh conditions, resist chemotherapy and radiation, and fuel tumor progression. Mitochondria-generated reactive oxygen species (ROS) act as signaling molecules to support self-renewal, tumorigenesis, and DNA repair, further strengthening TiSC resilience to therapy. Additionally, TiSCs evade apoptosis by tightly regulating cytochrome c release and caspase activation, making them more resistant to therapy-induced cell death. They also hijack mitochondria from immune cells, such as T cells and macrophages, impairing their function and promoting an immunosuppressive TME. Mitochondria with mtDNA mutations can be transferred to tumor-infiltrating lymphocytes (TILs), further dampening immune responses. Targeting mitochondrial pathways—including OXPHOS inhibition, ROS modulation, and disrupting mitochondrial transfer—could restore immune function, weaken TiSC defenses, and reduce tumor relapse. These therapeutic strategies represent promising avenues for improving cancer treatment outcomes.