The prebiotic activity of a commercially available oat product and a novel oat ingredient, at similar β-glucan loads, was tested using a validated in vitro gut model (M-SHIME®). The novelo at ingredient was tested further at lower β-glucan loads in vitro, while the commercially available oat product was assessed in a randomised, single-blind, placebo-controlled, and cross-over human study. Both approaches focused on healthy individuals with mild hypercholesterolemia. In vitro analysis revealed that both oat products strongly stimulated Lactobacillaceae and Bifidobacteriaceae in the intestinal lumen and the simulated mucus layer, and corresponded with enhanced levels of acetate and lactate with cross-feeding interactions leading to an associated increase in propionate and butyrate production. The in vitro prebiotic activity of the novel oat ingredient remained at lowerβ-glucan levels, indicating the prebiotic potential of the novel oat product.