EpiCor, derived from Saccharomyces cerevisiae, has been shown to have immunomodulating properties in human clinical trials and in vitro. However, the underlying mechanisms behind its immune protection via the gut remain largely unknown. Therefore, the aim of this study was to use an integrated in vitro approach to evaluate the metabolism of EpiCor by the intestinal microflora, its modulating effect on the gut microbiota, and its anti-inflammatory activity on human-derived cell lines. Using the SHIME model, in combination with a mucus adhesion assay, has shown that low doses of EpiCor have a prebiotic-like modulatory effect on the luminal- and mucosa-associated microbiota. These include gradual changes in general community structure, reduction of potential pathogens, quantitative increase in lactobacilli, and qualitative modulation of bifidobacteria. Moreover, by combination of the SHIME with Caco-2 cells and Caco-2/THP1 cocultures, a significant decrease in proinflammatory cytokines was observed at the end of the treatment period.