Dietary fibers can alter microbial metabolic output in support of healthy immune function, however, the impact of distinct fiber sources and immunomodulatory effects beyond short-chain fatty acid production are underexplored. In an effort to discern the effects of diverse fibers on host immunity, we employed five distinct rodent diets with varying fiber content and source in specific-pathogen-free, gnotobiotic (containing a 14-member synthetic human gut microbiota), and germ-free mice. Broad-scale metabolomic analysis of cecal contents showed a reduction in microbiota-produced B vitamins during fiber deprivation. Fiber deprivation shifted the ratio of transcripts corresponding to bacterial enzymes involved in B vitamin synthesis versus downstream utilization, resulting in a net reduction of host-available B vitamins. Broad immunophenotyping identified that the local gut effector immune populations and activated T cells accumulate in a microbiota-dependent manner. Supplementation with the prebiotic inulin recovered the availability of microbial-produced B vitamin and restored immune homeostasis. Our findings highlight the biotechnological potential of defined fiber polysaccharides to boost microbiota-derived B vitamin availability to the host and regulate local innate and adaptive immune populations, which may have critical implications for immune homeostasis in a variety of diseases.