OBJECTIVE: To investigate the effect of 5 newly developed maize-based fibers on the activity and composition of the microbiota in the colon. The fibers tested were glucose-based and had variable structures, including 2 resistant starch preparations, soluble corn fiber, pullulan, and soluble fiber dextrin. METHODS: The fibers were predigested, mono- and disaccharides were removed, and the residual polymer was used to assess the production of microbial metabolites and changes in composition of the microbiota using a dynamic, validated, in vitro model of the large intestine. RESULTS: Microbial metabolite analysis showed an increase in short-chain fatty acids for all fibers, with varying levels of butyrate production for each fiber. The greatest increase of butyrate, both in terms of absolute amounts and as a proportion of total short-chain fatty acids, was observed for pullulan. All fibers also reduced toxic metabolites from protein fermentation compared to the poorly fermentable control (cellulose). Microbial composition was assessed using a micro-array platform. All fibers showed increases of bifidobacteria and some Lactobacillus species, although different species were stimulated by different fibers. Pullulan showed the largest increase of bifidobacteria. CONCLUSIONS: All fibers showed prebiotic activity in terms of increases in growth and/or activity of beneficial microbes. In addition, compared to the control, health-promoting metabolites were produced in higher amounts, while putrefactive metabolites were reduced for all fibers. The importance of the findings lies in the fact that the newly developed, maize-based fibers shift the intestinal environment to a healthier milieu, with increased health-promoting metabolites and health-beneficial microbes.