Colonic fermentation of dietary fiber produces carboxylic acids and may stimulate the growth of beneficial bacteria. This study investigated how byproducts of wheat processing (distillers' grains and two fractions from the wet fractionation to starch and gluten, one of which was treated with xylanase) affect the composition of the cecal microbiota and the formation of carboxylic acids in rats. Differences were mostly found between diets based on supernatants and pellets, rather than between fiber sources. Cecal pools and levels of most carboxylic acids in portal blood were higher for rats fed the supernatant diets, while cecal pH and ratios of acetic to propionic acid in portal blood were lower. The diet based on supernatant from distillers' grains gave the highest level of bifidobacteria. Molecular weight and solubility are easier to modify with technological processes, which provides an opportunity to optimize these properties in the development of health products.