Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria‐endoplasmic reticulum (ER) contact sites called MAMs (mitochondria‐associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca²⁺ overload caused by Ca²⁺ release from ER via inositol‐3‐phosphate receptors (IP3Rs) and by Ca²⁺ entry through plasma membrane. This results in Ca²⁺‐dependent calpain activation, mitochondrial depolarization and cell death. The HK2‐targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti‐neoplastic strategy.