Arabinoxylan oligosaccharides (AXOS) are studied as food compounds with prebiotic potential. Here, the impact of consumption of breads with in situ-produced AXOS on intestinal fermentation and overall gastrointestinal characteristics was evaluated in a completely randomized, double-blind, controlled, cross-over study. Twenty-seven healthy volunteers consumed 180 g of wheat/rye bread with or without in situ-produced AXOS (WR(+) and WR(-), respectively) daily for 3 wk. Consumption of WR(+) corresponded to an AXOS intake of ~2.14 g/d. Refined wheat flour bread without AXOS (W(-)) (180 g/d) was provided during the 3-wk run-in and wash-out periods. At the end of each treatment period, participants collected urine for 48 h as well as a feces sample. Additionally, all participants completed a questionnaire about stool characteristics and gastrointestinal symptoms during the last week of each period. Urinary phenol and p-cresol excretions were significantly lower after WR(+) intake compared to WR(-). Consumption of WR(+) significantly increased fecal total SCFA concentrations compared to intake of W(-). The effect of WR(+) intake was most pronounced on butyrate, with levels 70% higher than after consumption of W(-) in the run-in or wash-out period. Consumption of WR(+) tended to selectively increase the fecal levels of bifidobacteria (P = 0.06) relative to consumption of W(-). Stool frequency increased significantly after intake of WR(+) compared to WR(-). In conclusion, consumption of breads with in situ-produced AXOS may favorably modulate intestinal fermentation and overall gastrointestinal properties in healthy humans.