Bone metastasis represents one of the most aggressive and painful late-stage manifestations of prostate cancer. Current treatments, primarily docetaxel-based chemotherapy with or without radiation, provide only temporary benefit, as relapses driven by therapy-resistant cancer stem-like cells (CSCs) are common. These CSCs are maintained by active gene networks that promote self-renewal through symmetrical division while retaining the ability for asymmetrical division and differentiation. However, the molecular mechanisms that govern CSC differentiation and therapy resistance remain poorly understood. To elucidate transcriptomic signatures associated with CSC differentiation, we profiled gene expression in androgen-repressive human prostate cancer C4-2B cells. CSC-enriched and non-CSC fractions were isolated using magnetic bead separation with CD133 antibody, followed by fluorescence-activated cell sorting and ALDH1 assay to identify the CD133⁺/ALDH1 high and CD133⁻/ALDH1 low populations. RNA sequencing revealed 33,676 expressed genes, of which 615 were significantly differentially expressed (p < 0.05). After false discovery rate correction, 56 genes remained significant (q < 0.05). Pathway analysis using QIAGEN Ingenuity Pathway identified activation of the sirtuin signaling pathway as a key feature in the CSC fraction, alongside enrichment of oxidative phosphorylation (OXPHOS), mitochondrial dysfunction, B cell receptor, CXCR4, Th2, IL17A, and TNFR2 signaling pathways. Transcript profiling of the sirtuin gene family (SIRT1-SIRT7) revealed marked upregulation of mitochondrial sirtuins: SIRT3, SIRT4, and SIRT5 in the CD133⁺/ALDH1 high population, consistent with enhanced OXPHOS activity relative to the CD133⁻/ALDH1 low cells. Network analysis further demonstrated that the SIRT3-SIRT4-SIRT5 interactome integrates into the NAD⁺ metabolic framework, connecting key enzymes involved in NAD⁺ synthesis and utilization, including NADSYN1, NMNAT1/2, NADK2, BST1, NNMT, and ENPP3. Notably, SIRT3 emerged as the central regulatory hub, coordinating interactions with SIRT4 and SIRT5 to sustain oxidative metabolism and suppress reactive oxygen species accumulation. This coordination promotes metabolic flexibility, redox balance, and resistance to differentiation cues which are hallmarks of CSC persistence and therapeutic resistance. Collectively, these findings identify mitochondrial sirtuins, particularly SIRT3, as key regulators of metabolic reprogramming and redox homeostasis in castration-resistant CSCs. Targeting mitochondrial sirtuin signaling, alone or with chemotherapy, represents a promising strategy to eliminate CSCs and prevent prostate cancer progression and bone metastasis.