Dietary fiber is an important nutrient for improving human health and controlling calorie intake, and is used to produce functional foods. In this study, insoluble dietary fiber (IDF) from three sources (enoki mushrooms, carrots, and oats) was characterized and their hypoglycemic and hypolipidemic effects were determined with in vitro and in vivo models. The results of Scanning electron microscopy (SEM) showed that the IDF from the three sources have different morphologies. The Fourier transform infrared spectroscopy (FT-IR) results showed that the IDF samples from the three sources have similar active groups, but the X-ray diffraction (XRD) and thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) results indicated that oat IDF mainly contained cellulose, and enoki mushroom IDF and carrot IDF contained hemicelluloses and cellulose. Among three IDF, carrot IDF had stronger water holding capacity, swelling capacity, and adsorption capacity of oil and cholate; enoki mushroom IDF had stronger glucose adsorption capacity and the ability to inhibit fat digestion; while oat IDF had stronger cholesterol adsorption capacity. None of the three IDF showed significant inhibition on starch digestion. Results from mouse feeding studies showed that IDF from three sources all improved glucose tolerance and inhibited the rise of blood lipid after the fat loading. Thus, this study demonstrated the functional significance of the IDF from three sources, which provides a reference for their application in functional food products aiming at maintaining healthy glucose and blood lipid levels.