AbstractWe present the design and synthesis of a small library of substituted biguanidium salts and their capacity to inhibit the growth of pancreatic cancer cells. We first present their in vitro and membrane activity, before we address their mechanism of action in living cells and in vivo activity. We show that phenylethynyl biguanidium salts possess higher ability to cross hydrophobic barriers, improve mitochondrial accumulation and anticancer activity. Mechanistically, the most active compound, 1b, like metformin, activated AMPK, decreased the NAD⁺/NADH ratio and mitochondrial respiration, but at 800-fold lower concentration. In vivo studies show that compound 1b significantly inhibits the growth of pancreatic cancer xenografts in mice, while biguanides currently in clinical trials had little activity.