G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na(+), K(+), Mg(2+) and Ca(2+), promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca(2+) has the strongest stabilizing effect, followed by K(+), Mg(2+), and Na(+) in a decreasing order. The binding of K(+) to G-triplexes is accompanied by exothermic heats, and the binding of Ca(2+) with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K(+) or Ca(2+). These observations imply that stable G-triplexes may be formed under physiological conditions.