alpha-Glucosidase has emerged as an important target for the therapy of type 2 diabetes mellitus. In our previous study, the natural product salvianolic acid C was found to present a potent alpha-glucosidase inhibitory activity, whereas the interaction mechanisms are still not clear. Herein, an integrated approach consisting of inhibition kinetics, multi-spectroscopic methods and molecular modelling was applied to investigate the interaction mechanism between salvianolic acid C and alpha-glucosidase. As a result, salvianolic acid C showed a potent alpha-glucosidase inhibitory activity with IC50 of 3.03 +/- 0.27 mu M in a mixed-competitive manner. The activity of alpha-glucosidase was significantly inactivated by salvianolic acid C with the first-order reaction. The fluorescence assay suggested that salvianolic acid C presented a strong fluorescence quenching effect in a static quenching mechanism and the hydrophobic interaction was demonstrated to be the predominant driving force. The synchronous fluorescence and FT-IR spectra indicated that salvianolic acid C could induce the rearrangement and conformational changes of the enzyme. Additionally, molecular modelling studies further validated that salvianolic acid C could bind to the active center of a-glucosidase through hydrophobic interaction and hydrogen bonds. Such findings provide some useful information for us to discover novel a-glucosidase inhibitors based on salvianolic acid C.