Diet is an important risk factor for colorectal cancer (CRC) and several dietary constituents implicated in CRC are modified by gut microbial metabolism. Microbial fermentation of dietary fiber produces short chain fatty acids, e.g., acetate, propionate, and butyrate. Dietary fiber has been shown to reduce colon tumors in animal models and, in vitro, butyrate influences cellular pathways important to cancer risk. Further, work from our group suggests that the combined effects of butyrate and omega-3 polyunsaturated fatty acids (n-3 PUFA) may enhance the chemopreventive potential of these dietary constituents. We postulate that the relatively low intakes of n-3 PUFA and fiber in Western populations and the failure to address interactions between these dietary components may explain why chemoprotective effects of n-3 PUFA and fermentable fibers have not been detected consistently in prospective cohort studies. We hypothesize that the combined effects of longchain n-3 PUFA supplementation and supplemental highly fermentable fiber will alter critical pathways important to CRC prevention, particularly intrinsic mitochondrial-mediated programmed cell death resulting from the accumulation of lipid reactive oxygen species (ferroptosis), cell proliferation, and the eicosanoid/inflammation pathways. We propose a randomized, controlled crossover pilot study in 30 healthy men and women (50-75 y) to compare supplemental soluble fiber (35 g/d) + supplemental n-3 PUFA (6 g/d EPA+DHA), in quantities mirroring mean daily intakes associated with lower CRC risk, with a maltodextrin and corn oil control. Stool samples will be collected at the beginning, middle (day 15), and end of each of the two 30-day intervention periods. Using a novel, cost-effective, non-invasive approach, we will evaluate differences in global gene expression signatures in the stool exfoliome (i.e., sloughed colonic epithelial cells in stool) using RNA-Seq. We will focus on pathways related to colonic cell proliferation and apoptosis/ferroptosis, cell phenotype, and inflammatory response. Further, we will evaluate changes in gut microbial functional genes involved in butyrate production using droplet digital PCR (ddPCR). The collection of multiple samples over the intervention periods will provide a critical measure of longitudinal changes in response to the supplemental n-3 PUFA and fermentable fiber. This proposed pilot human mechanistic study will be the first in humans to integrate and characterize the relationships between n-3 PUFA and fermentable fiber exposure that modulate programmed cell death and other CRC-related cell-signaling pathways through metabolic activities of the gut microbiome. Results of this controlled intervention will help to translate the current mechanistic knowledge from preclinical animal models to humans and to de-risk and inform approaches for CRC prevention. Interrogating the stool exfoliome is a novel, cost-effective, non-invasive approach to studying effects of interventions on the human gut.