Osmolytes are known to stabilize proteins under stress conditions. Thermal denaturation studies on globular proteins (β-lactoglobulin, cytochrome c, myoglobin, α-chymotrypsin) in the presence of ethylene glycol (EG), a polyol class of osmolyte, demonstrate a unique property of EG. EG stabilizes proteins against cold denaturation and destabilizes them during heat-induced denaturation. Further, chemical denaturation experiments performed at room temperature and at a sub-zero temperature (-10 °C) show that EG stabilizes the proteins at subzero temperature but destabilizes them at room temperature. The experiments carried out in the presence of glycerol, however, showed that glycerol stabilizes proteins against all of the denaturing conditions. This differential effect has not been reported for any other polyol class of osmolyte and might be specific to EG. Moreover, molecular dynamics simulations of all of the four proteins were carried out at three different temperatures, 240, 300, and 340 K, in the absence and presence of EG (20 and 40%). The results suggest that EG preferably accumulates around the hydrophobic residues and reduces the hydrophobic hydration of the proteins at a low temperature leading to stabilization of the proteins. At 340 K, the preferential hydration of the proteins is significantly reduced and the preferential binding of EG destabilizes the proteins like common denaturants.