TFE, 2,2,2-trifluoroethanol, and guanidinium (Gdm+) are two typical osmolytes. A great number of experimental and theoretical studies have shown that TFE and Gdm+ can accumulate on protein surface and thus exert their effects on protein structure in their respective solutions. Their accumulation manners are, however, different: the hydrophobic property of TFE makes its accumulation more preferential around hydrophobic side chains than other types whereas Gdm+ prefers to stack strongly against the planar side chains but only weakly binds to the hydrophobic groups. The present molecular dynamics simulation study shows a novel test to investigate the combined effects of TFE and Gdm+ on protein structure in mixed guanidinium/TFE solution. The results indicate that the accumulation of TFE is more competitive than Gdm+ in either GdmSCN/TFE or GdmCl/TFE solution. The preceding accumulation of TFE around protein surface limits the approach of Gdm+ and water to protein. As a result, the hydrogen bonding between Gdm+ and water to protein is highly forbidden and the secondary structure stability of protein is strongly enhanced. In contrast, without the presence of TFE, the protein structure is largely denatured in similarly concentrated GdmSCN or GdmCl solution. Proteins 2014; 82:944–953. © 2013 Wiley Periodicals, Inc.