Background/Objectives <p>The gut-microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) has been linked to cardiovascular disease (CVD) risk, while dietary fiber is associated with reduced CVD risk and improved gut health. Considering these associations, we conducted a randomized, double-blind, pilot study to investigate the influence of fiber supplementation on intestinal TMAO formation after beef consumption.</p> Subjects/Methods <p>13 volunteers underwent a two-week dietary fiber and placebo intervention. We assessed the effect of fiber supplementation on the gut microbiota and gene abundance of the enzyme cutC, a key enzyme for microbial TMA formation, a precursor for TMAO. We measured the TMAO response following beef consumption after the two-week intervention. We also examined the role of hepatic enzyme FMO3 on TMAO plasma levels.</p> Results <p>Although overall TMAO production did not change between the dietary fiber and placebo group (<i>p</i>-value = 0.26, 95% CI), subgroup analysis revealed that fiber supplementation attenuated TMAO formation following beef intake in participants with lower habitual meat consumption ( &lt;3 times/week, <i>p</i>-value = 0.029, 95% CI). Furthermore, fiber intervention significantly downregulated microbial cutC gene abundance (<i>p</i> = 0.034, 95% CI), suggesting a mechanism by which fiber might reduce plasma TMAO levels. While dietary fiber intervention did not alter TMAO production across all participants, it showed a potential effect in individuals with lower habitual meat intake. The observed downregulation of cutC gene abundance suggests a mechanism for the beneficial impact of fiber on TMAO formation.</p> Conclusion <p>These findings support the role of a high-fiber, low-meat diet as a promising strategy to mitigate TMAO-related CVD risk.</p> <p></p>