There have been many reports concerning the role of dietary fiber in lowering postprandial serum glucose, and the main mechanism was regarded as the viscosity of different dietary fibers in hampering diffusion of glucose and postponing absorption and digestion of carbohydrates. In this paper, two kinds of water-insoluble dietary fibers, water-insoluble dietary fiber of wheat bran and enzyme-resistant starch of maize amylose, and four kinds of water-soluble dietary fibers, water-soluble dietary fiber of wheat bran, carboxymethyl cellulose, guar gum, and xanthan gum, were used to investigate their postprandial serum glucose lowering mechanism in vitro. The results showed that these dietary fibers lowered postprandial serum glucose levels at least by three mechanisms. First, dietary fibers increase the viscosity of small intestine juice and hinder diffusion of glucose; second, they bind glucose and decrease the concentration of available glucose in the small intestine; and, third, they retard alpha-amylase action through capsuling starch and the enzyme and might directly inhibit the enzyme. All of these decreased the absorption rate of glucose and the concentration of postprandial serum glucose.