Researchers have reported enzymatic reactions performed in ionic liquids (ILs). Currently very little is known about the behavior of biomolecules in ILs in comparison to their better understood aqueous behavior. We report the temperature-dependent behavior of human serum albumin (HSA) that is site-selectively labeled at cysteine-34 (located in loop 1 of domain I) with a single fluorescent reporter molecule (acrylodan, Ac) when it is dissolved in phosphate buffered saline (PBS) or one of three ILs. In PBS, the Ac reporter motion is always coupled to the global HSA protein motion. In the ILs, loop 1 of domain I appears to be almost completely decoupled from the HSA global motion. As temperature increases, neighboring nonpolar amino acid residues and/or IL components apparently become strongly associated with the Ac rotating body. These results show that protein structure and dynamics in an IL can markedly deviate from that which exists in aqueous media.