The present study investigated the antioxidant activities and hypocholesterolemic properties of soluble dietary fiber (SDF) obtained from Qing Ke brewer’s spent grain using Caco-2 cells. SDF simultaneously increased the activities and mRNA expression levels of the phase II detoxifying/antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidise (GPx). The expression levels of the intestinal genes for cholesterol efflux, ABCA1, ABCG5, and LXRα, were up-regulated by treatment with SDF. Moreover, SDF decreased the transcriptional levels of genes related to cholesterol absorption and synthesis in Caco-2 cells. The results suggested that hypocholesterolemic effects of SDF may be due, at least in part, to its antioxidant activities.