Investigation of interaction between rotenone (Rot) and human serum albumin (HSA) is provided. Fluorescence, UV-visible absorption, circular dichroism (CD), and molecular docking methods have been utilized to study the interaction. The static fluorescence quenching was observed and the binding site of Rot-HSA was shown near the warfarin site. The association for Rot and HSA was spontaneous process driven by enthalpy and the dominant driving force was hydrogen bonding interaction. The result was accordant with the molecular docking result. Furthermore, the microenvironment of tyrosine residue was influenced by the binding of rotenone via the synchronous fluorescence spectra. However, Rot had little effect on the conformation of HSA, which supported by the CD method and 3D-fluorescence spectra. Taking all of results into consideration, we conclude Rot changes the conformation of HSA slightly. (C) 2013 Elsevier Ltd. All rights reserved.