Yeast, fungal, and dietary β-glucans have immune-modulating effects in vitro and in vivo, as thought, mainly by affecting leukocytes; however, effects of oat β-glucan on enterocytes have never been studied. As recognized, supplying oat β-glucans as such to cells in culture directly is difficult because of solubility problems. Therefore, six ileostomic patients consumed, in random order, a control diet or an oat β-glucan enriched diet (5 g) and from the collected ileostomic content, fecal water was prepared and added to two small intestinal cell lines (INT407, Caco-2) and two colon cell lines (HT29, T84) together with a cytokine cocktail (IL-1β + INFγ + TNFα). Several parameters reflecting immune-modulation were measured. As compared to placebo fecal water, β-glucan enriched fecal water significantly increased IL-8 production in HT29 (5.0%; p = 0.046) and INT407 cells (22.0%; p = 0.028). Intercellular adhesion molecule (ICAM)-1 expression increased in T84 (11.0%; p = 0.028) and Caco-2 cells (20.4%; p = 0.075). These immune-stimulating effects were confirmed by enhancement of inflammatory expression profiles, as determined with an antibody array. Our findings show immune enhancement by fecal water from ileostomic patients consuming oat β-glucan both in small intestinal and colon cell lines after stimulation, which is in agreement with documented effects in leukocytes. Whether these immune-stimulating effects on enterocytes contribute to the enhanced protection of the host against invading pathogens as observed both in animals and in humans, as well as the underlying mechanism, needs further evaluation.