The binding reaction of colchicine with human serum albumin (HSA) was studied by UV-Vis absorption, fluorescence and circular dichroism spectrometry. The results indicated that colchicine led to the increase in UV absorption and the quenching of intrinsic fluorescence of HSA. As the temperature increased, the quenching constant Ksv decreased. The binding constants and the numbers of the binding sites of the interaction between colchicine and HSA at different temperatures were obtained. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS), were calculated to be -11.66 kJ x mol(-1) and 51.507 J(mol x K)(-1) respectively according to Van't Hoff equation, which suggested that the main binding force between colchicine and HSA was static interaction. The protein conformation was altered (CD date) with decreasing of alpha-helices in the presence of colchicine. The results showed that the quenching mechanism of the combination of colchicine with human serum albumin was a static quenching procedure.