BACKGROUND AND AIMS: Antioxidants, including vitamin E (VE) and grape seed extract (GSE), as anti-aging supplementation have been widely used to improve human health. The gut microbiota plays a crucial role in health and affects the treatment effect of various interventions. However, the role of gut microbiota in VE remains unclear. This study aimed to assess the longitudinal impact of VE treatment on body health and the gut microbiota. METHODS AND RESULTS: A randomized controlled trial was conducted with 90 healthy individuals. The participants were randomly assigned to three groups: a treatment group receiving VE, another antioxidant treatment group receiving GSE, and a control group receiving a placebo. We found that VE ameliorated blood cholesterol levels by reducing the levels of low-density lipoprotein cholesterol (LDL-C) in healthy volunteers. After the intervention, there was an increase in the relative abundance of short-chain fatty acid (SCFA)-producing bacteria and bile acid metabolizers. Specifically, the abundances of Lachnospira sp. and Faecalibacterium spp. increased in the VE. Interestingly, the gut microbiota of poor responders harbored a greater proportion of disease-associated bacterial species. CONCLUSIONS: VE could promote health by lowering LDL-C, partly and indirectly by affecting gut bacteria with the ability to produce SCFAs or metabolize bile acids. REGISTRATION NUMBER FOR CLINICAL TRIALS: The clinical trial was registered on August 28, 2021. Registration number was ChiCTR2100050567 (https://www.chictr.org.cn).