The gut microbiome plays a key role in regulating metabolic health, and dietary interventions can beneficially reshape its composition and function, contributing to weight loss and improved metabolic outcomes. However, weight regain—often observed after 6 months of active intervention—may be partly driven by a reversion of the microbiome toward its baseline state. Autologous fecal microbiota transplantation (aFMT), which uses an individual’s own stool collected during a metabolically healthier state, offers a minimal-risk strategy that may preserve diet-induced microbial shifts and reduce weight regain. In the DIRECT-PLUS aFMT Trial, we examined the effects of randomized aFMT versus placebo on gut microbiome—particularly strain-level changes—and their role in mitigating weight regain in 90 adults. Participants who lost ≥3.5% of body weight during a 6-month dietary intervention (Healthy Dietary Guidelines [HDG], Mediterranean diet [MED], or Green Mediterranean diet [GreenMED]) provided fecal samples that were processed into oral capsules. During the 6-month follow-up, aFMT recipients experienced significantly less weight regain compared to placebo, especially when combined with GreenMED. In the overall study population, aFMT did not differ from placebo in preserving prior diet-induced microbial strain-level changes. However, when combined with the GreenMED diet, aFMT effectively preserved these strain-level changes during the follow-up period. Notably, enhanced persistence of specific strains, such as Ruminococcus lactaris, Bifidobacterium longum, and Blautia obeum, was observed in the aFMT group, particularly when combined with GreenMED. This may reflect the potential of aFMT to enhance the colonization and persistence of microbial strains capable of degrading complex polysaccharides and metabolizing polyphenols in the gut. aFMT also preserved diet-induced changes in fecal metabolites, such as increased levels of pheophorbide A, serotonin, and N-methylpipecolate, alongside decreased level of 8-hydroxyoctanoate, glycophosphoinositol, and N-palmitoyl-heptadecasphingosine. These metabolomic profiles represent a gut environment favoring enhanced plant-food metabolism and gut-brain signaling, and reduced oxidative stress. Critically, multi-omic signatures, including shifts in species abundance, dominant strain types, and metabolite profiles, effectively distinguished participants who regained body weight from those who maintained weight after six months of dietary intervention, with classification AUCs exceeding 85%. Together, these findings suggest that aFMT, particularly when paired with a polyphenol-rich dietary pattern, induces strain-level shifts and functional changes in the gut microbiome that help reduce weight regain and improve metabolic risk profiles. Harnessing an individual’s own microbiome as a therapeutic intervention offers a novel, personalized approach to support long-term weight maintenance and metabolic health.