Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.03 was obtained after addition of 1% (w/v) WU-AX to a pH-controlled stirred anaerobic fermentation vessel. Pretreatment of the WU-AX with endo-β-1,4-xylanase resulted in significantly higher PI value (3.48) indicating that pretreatment provided oligomers that were better utilised by the gut bacteria. The extracellular hydrolytic enzymes xylanase and ferulic acid esterase are both required for bacterial metabolism of WU-AX and both activities were present in supernatants derived from the mixed batch cultures. Addition of the WU-AX substrates to the batch cultures produced several fold increases of bacterial synthesis of both enzymes, and these increases were greater when the WU-AX substrate was pretreated with xylanase.