This study investigated the effects of fermentable dietary fiber derived from high-amylose wheat (HAW) flour on the intestinal environment using an in vitro fecal fermentation assay and a randomized, double-blind, parallel-group clinical trial. Digested HAW flour was fractionated into total dietary fiber (TDF), resistant starch (RS), and non-RS dietary fiber (DF-RS) fractions. Fecal culture tests were used to quantify short-chain fatty acid (SCFA) production and microbiota composition after cultivation. In the randomized, double-blind, parallel-group trial, 76 healthy adults consumed HAW-containing food (dietary fiber: 5.5 g/day, RS: 2.9 g/day) or control food (dietary fiber: 0.7 g/day, RS: n.d.) for 2 weeks. Both RS and DF-RS increased SCFA production, with TDF having even stronger effects, suggesting enhanced fermentability in the presence of multiple types of fermentable dietary fibers. In the human trial, HAW-containing food intake did not significantly alter bowel movement frequency compared with the control. However, HAW-containing food consumption significantly reduced the levels of p-cresol, a representative gut-derived proteolytic metabolite linked to intestinal dysbiosis. No significant differences were observed in other secondary endpoints. Intake of HAW-derived foods appears to promote SCFA production and improve the intestinal environment by reducing p-cresol accumulation. Overall, these results highlight HAW flour as a practical prebiotic ingredient that helps support gut health.