3-mercaptopyruvate sulfurtransferase (3-MST) is involved in hydrogen sulfide production and cellular metabolism. Here, we investigated its role in colorectal cancer stem cells (CSCs), a therapy-resistant population with reduced bioenergetic activity. Proteomic analyses were performed in HCT116 wild-type cells (WT), CSCs and Rescue cells generated by CSC re-differentiation. Inhibition of 3-MST induced a marked bioenergetic impairment and promoted CSC death. DIA-based proteomics revealed significant remodeling of metabolic pathways, including glycolysis, TCA cycle, and lipid metabolism, along with activation of stress and cell death pathways. This dataset provides a resource to study the metabolic role of 3-MST in CSCs.