The interaction between imidacloprid (IMI) and human serum albumin (HSA) was investigated using fluorescence and UV/vis absorption spectroscopy. The experimental results showed that the fluorescence quenching of HSA by IMI was a result of the formation of IMI-HSA complex; static quenching was confirmed to result in the fluorescence quenching. The apparent binding constant K(A) between IMI and HSA at three differences were obtained to be 1.51 x 10(4), 1.58 x 10(4), and 2.19 x 10(4) L mol(-1), respectively. The thermodynamic parameters, DeltaH degrees and DeltaS degrees were estimated to be 28.44 kJ mol(-1), 174.76 J mol(-1) K(-1) according to the van't Hoff equation. Hydrophobic interactions played a major role in stabilizing the complex. The distance r between donor (HSA) and acceptor (IMI) was obtained according to fluorescence resonance energy transfer. The effect of IMI on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy CD and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.