Astilbin is a dihydroflavonol that is widely distributed in many foods and plants. Our previous study showed astilbin could significantly decrease the body weight gain and intraperitoneal adipose tissue weight, as well as triglyceride content in serum. In this paper, we studied the inhibitory activity of astilbin on pancreatic lipase. The results showed that astilbin had inhibitory effect on pancreatic lipase with IC50 of 105 μM and maximum inhibition rate of 56.3%. The inhibitory effect increased with the interaction time between astilbin and pancreatic lipase. Enzyme kinetics study showed that astilbin is a noncompetitive inhibitor of pancreatic lipase, which does not change its affinity with the substrate, but decrease its maximum reaction rate. The fluorescence spectra showed that astilbin could quench the endogenous fluorescence of pancreatic lipase, and make its maximum emission wavelength red shift, their binding constant (lgK_a) was 5.50 and the binding-site number was 1.17.