The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM Es with the same cation 1-butyl-3-methylimidazolium (Bmim(+)) but different anions (BF4-, NO3-, Cl-, and Br-) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of Es in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF4-, NO3-, Cl-, and Br-) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim(+) cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of Es increases with increasing alkyl chain length of imidazolium cation of Es and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.