Exploring the interaction between rotenone and human serum albumin

•The fluorescence quenching mechanism of HSA induced by rotenone is static quenching.•The association for rotenone and HSA is a spontaneous process driven by enthalpy.•Hydrogen bond is the dominant driving force.•Rotenone binding to site I of HSA influences the microenvironment of tyrosine residue.•The binding of rotenone affects the conformation of HSA slightly.