Abstract Body (Do not enter title and authors here): Resistant starch (RS), a major category of dietary fiber, promotes healthy lipid and glucose metabolism. These effects are partly mediated by gut microbes. RS is known to influence microbial bile acid metabolism and, in turn, can lower serum cholesterol. We hypothesized that RS may promote another microbial process that affects serum cholesterol levels – the cholesterol:coprostanol transformation pathway, which has not been fully characterized. Individuals who harbor gut microbes encoding a recently identified gene in the pathway, intestinal sterol metabolism A (ismA), can transform cholesterol to less absorbable coprostanol. Individuals with microbial ismA genes have lower serum cholesterol than people without microbial ismA genes. We conducted a seven-week crossover study with 59 individuals completing three treatments: dietary supplementation with RS type 2, RS type 4, and a digestible starch, for ten days each with five-day washout periods in between. We collected fecal samples at the beginning and end of each treatment and extracted DNA to generate a shotgun metagenomics dataset from the gut microbiomes. We also measured the concentrations of bile acids and the sterols, cholesterol and coprostanol, in the stool. RS supplementation caused interindividual variability in gut microbiome response. Also, during the RS type 2 treatment, we observed a decrease in taurocholic acid, a bile acid that may promote colon cancer (q < 0.05). RS supplementation did not universally affect sterol levels. However, we observed consistent correlations between the abundances of some microbes, or species level genome bins (SGBs), and sterol concentrations longitudinally within subjects. We identified 24 SGBs, including three Oscillibacter species, which were negatively correlated with cholesterol and positively correlated with coprostanol (Spearman's rho ≥ 0.4). In addition to its ability to transform cholesterol, Oscillibacter has previously been implicated as benefiting lipid metabolism and cardiovascular health. We also identified 12 unclassified SGBs that may be involved in the transformation of cholesterol to coprostanol in the human gut. These SGBs may represent previously undiscovered species that participate in the cholesterol:coprostanol transformation pathway, and these species may possess unknown genes in the pathway. Comprehensive characterization of this pathway could lead to novel microbiome-based therapeutics to treat hypercholesterolemia.