Deoxynivalenol (DON), a highly prevalent mycotoxin food contaminant, is known to have immunotoxic effects. In the current study, the potential of dietary interventions with specific mixtures of trans-galactosyl-oligosaccharides (TOS) to alleviate these effects were assessed in a murine influenza vaccination model. Vaccine-specific immune responses were measured in C57Bl/6JOlaHsd mice fed diets containing DON, TOS or a combination, starting 2 weeks before the first vaccination. The direct effects of TOS and its main oligosaccharide, 3′-galactosyl-lactose (3′-GL), on DON-induced damage were studied in Caco-2 cells, as an in vitro model of the intestinal epithelial barrier. Exposure to DON significantly reduced vaccine-specific immune responses and the percentages of Tbet+ Th1 cells and B cells in the spleen. DON significantly altered epithelial structure and integrity in the ileum and reduced the SCFA levels in the cecum. Adding TOS into DON-containing diets significantly improved vaccine-specific immune responses, restored the immune cell balance in the spleen and increased SCFA concentrations in the cecum. Incubating Caco-2 cells with TOS and 3′-GL in vitro further confirmed their protective effects against DON-induced barrier disruption, supporting immune modulation. Overall, dietary intervention with TOS can attenuate the adverse effects of DON on Th1-mediated immune responses and gut homeostasis. These beneficial properties might be linked to the high levels of 3′-GL in TOS.