Analogous to reported immunomodulatory effects of probiotics, this study was performed to analyse the immunomodulatory properties of prebiotic oligosaccharides that share chemical characteristics with human milk oligosaccharides. A mixture containing galacto- and fructo-oligosaccharides (GOS/FOS; ratio 9:1) was tested at dietary doses between 1% and 10% (w/w of total diet) in an influenza vaccination model, using 10 C56BL/6JolaHsd mice per group. The modulation of vaccine specific delayed-type hypersensitivity (DTH) responses was studied as a marker of T-helper 1 (Th1) immunity, as well as other immune parameters. GOS/FOS enhanced DTH responses dose-dependently (optimum at 5% w/w of total diet; 41.4+/-14.1% increased compared to controls, p<0.05). No significant changes were detected on splenocyte proliferation or vaccine-specific antibody concentrations. Simultaneously, GOS/FOS dose-dependently increased the proportion of faecal bifidobacteria and lactobacilli (maximal effect at 10% w/w of total diet; 16.8+/-2.4% and 5.8+/-1.3% increased compared to controls respectively, p<0.01 for both parameters). In a comparative experiment, GOS/FOS and FOS/inulin (both at 2% w/w of total diet) induced similar significant effects on the gut microbiota. In contrast to GOS/FOS, FOS/inulin did not enhance DTH responses, indicating that an increase in the proportions of bifidobacteria and lactobacilli is not sufficient for an immunomodulatory effect in this model. The use of GOS/FOS in dietary products might provide an opportunity to stimulate the adaptive immune response in a Th1-direction and subsequently inhibit infections and Th2-related immune disorders in humans, for instance allergies. Clinical studies are being performed to confirm this.