Metal cations are essential for the function of nucleic acids. Given their efficiency for DNA overwinding and the DNA hairpin folding/unfolding process coupled with DNA twist, we systematically investigated the effects of cation identities and increasing concentrations of NaCl, KCl, and LiCl from 50 mM to 300 mM on the folding/unfolding process by manipulating a DNA hairpin construct with magnetic tweezers. The critical forces and free energy of the unfolding transition of the DNA hairpin at a fixed concentration of KCl, NaCl, and LiCl followed the same ranking as