To unravel the underlying mechanisms that explain the positive effects of prefermented cereals on in vivo gastrointestinal (GI) architecture and function, an in vitro experiment using a human small intestinal epithelial cell model (Caco-2) was performed. A range of dilutions (0% to 10%) of the supernatants of three liquid experimental diets, as well as Na-lactate were used in an in vitro experiment to assess their effect on cellular growth, metabolism, differentiation and mucosal integrity using Caco-2. The experimental diets contained, in addition to a protein rich basal diet (60%), (1) a liquid control diet (C) containing 40% of a mixture of barley and wheat (ratio 3 : 1) or (2) a liquid diet (F) containing 40% prefermented barley and wheat or (3) C with the addition of the fermentation end-products (organic acids and ethanol) in concentrations similar to those in the fermented diet (FP). For F, the mixture of barley and wheat was fermented at 35°C for 48 h. Parallel to the in vitro experiment, 18 groups of eight weanling pigs were assigned to one of the experimental diets during a 14-day in vivo experiment. Each group was fed restrictively. The results of the in vitro experiment showed that the lowest dose of both F- and FP-supernatants had no clear effects on the cell proliferation, but incubation with 5% and 10% of the F- and FP-supernatants decreased the cell numbers at day 19. DNA, RNA, protein and glycoprotein synthesis in differentiated Caco-2 cells were stimulated by incubation with the lower concentrations (0.5% to 2.5%) of F- and FP-supernatants whereas the higher concentrations (5% and 10%) had no effect. Both the F- and FP-supernatants decreased the specific sucrase–isomaltase activity in a dose-dependent manner, but the effects on the specific aminopeptidase activities were less clear. Mucosal integrity initially decreased after incubation with the highest F- and FP-supernatants and started to recover between 24 and 48 h. The results of the in vivo experiment showed no dietary effects (P > 0.1) on GI morphology and brush-border enzyme activities at day 5 or at day 14. Time related changes in GI characteristics followed a normal pattern. In conclusion, the supernatants of diets containing either prefermented cereals or their fermentation end-products clearly modulate cellular growth, metabolism, differentiation and mucosal integrity in an in vitro model, although these effects were not observed in the in vivo characteristics measured in weanling pigs.