The effect of extrusion processing on water-soluble dietary fiber (SDF) and water-insoluble dietary fiber (IDF) of whole grain highland barley (HB) in terms of morphology, physicochemical and functional properties, and antioxidant activities was studied. Extrusion processing resulted in the more porous and loose structure of SDF and IDF, and significantly increased the specific surface area of SDF and IDF. The predominant monosaccharide in IDF and SDF was cellulose. Extrusion processing improved the water holding capacity of SDF and IDF by 23.9% and 40.4%, respectively. The glucose absorption capacity and glucose dialysis retardation index of IDF and SDF were further significantly improved by extrusion processing, indicating the great potential of glucose control in vitro of IDF and SDF. The cholesterol adsorption capacity, nitrite adsorption capacity, and cation exchange capacity of IDF and SDF were also significantly improved through extrusion processing. Extrusion processing significantly improved the antioxidant activities of IDF and SDF which reflected as the improved DPPH and ABTS⁺ scavenging activities. Taken together, extrusion processing improved the physicochemical and functional properties of dietary fiber of whole grain HB, as well as the antioxidant activities. This study showed the great potential of dietary fiber of whole grain HB in the applications of functional foods for improving the health benefits.