Strawberry is a rich source of soluble dietary fibers, mainly pectin. Previous studies have revealed the health promoting effects of whole strawberry via gut microbiota modulation possibly due to its dietary fiber content. This research delved into the multifaceted aspects of strawberry soluble fiber (SSF), aiming to unravel its prebiotic potential and its impact on gut microbial dynamics and intestinal health. Specific Aim 1 investigated the chemical composition of SSF, revealing a pectic polysaccharide structure through molecular weight distribution analysis, monosaccharide composition and FT-IR spectroscopy. Fermentation studies affirmed SSF's prebiotic potential, as evidenced by alterations in gut microbial communities and increased short-chain fatty acid (SCFA) production. Comparative analysis with citrus pectin suggested SSF's promising role in improving gut microbial profiles. Specific Aim 2 focused on isolating and characterizing human gut bacteria capable of fermenting SSF. Three distinct Bacteroides species, B. vulgatus, B. faecis, and B. xylanisolvens, exhibited enhanced growth in response to SSF, emphasizing its prebiotic properties. The study highlighted the potential synergy between SSF and specific bacterial strains, paving the way for a deeper understanding of SSF's positive impacts on gut health. In Specific Aim 3, an animal study explored the interplay between SSF, gut microbiota, and intestinal structure. SSF supplementation, alongside citrus pectin, demonstrates dose-dependent effects on jejunal villus height, jejunal V/C ratio, and SCFA profiles. Bile acid analysis revealed changes in primary and secondary bile acids, emphasizing the influence of SSF on host metabolism. Gut microbial community analysis at various taxonomic levels provided insights into the intricate relationships governing gut microbiota dynamics in response to SSF and bacterial treatments. Overall, this comprehensive investigation unveiled SSF as a potential food ingredient with prebiotic properties, offering valuable insights into its chemical composition, microbial modulation effects, and impacts on intestinal health. Further research regarding molecular mechanism of action is essential to solidify SSF's role as a prebiotic and its potential applications in promoting gut health.