OBJECTIVE To determine the longer-term effects of metformin treatment and behavioral weight loss on gut microbiota and short-chain fatty acids (SCFAs). RESEARCH DESIGN AND METHODS We conducted a 3-parallel-arm, randomized trial. We enrolled overweight/obese adults who had been treated for solid tumors but had no ongoing cancer treatment and randomized them (n = 121) to either 1) metformin (up to 2,000 mg), 2) coach-directed behavioral weight loss, or 3) self-directed care (control) for 12 months. We collected stool and serum at baseline (n = 114), 6 months (n = 109), and 12 months (n = 105). From stool, we extracted microbial DNA and conducted amplicon and metagenomic sequencing. We measured SCFAs and other biochemical parameters from fasting serum. RESULTS Of the 121 participants, 79% were female and 46% were Black, and the mean age was 60 years. Only metformin treatment significantly altered microbiota composition. Compared with control, metformin treatment increased amplicon sequence variants for Escherichia (confirmed as Escherichia coli by metagenomic sequencing) and Ruminococcus torques and decreased Intestinibacter bartlettii at both 6 and 12 months and decreased the genus Roseburia, including R. faecis and R. intestinalis, at 12 months. Effects were similar in comparison of the metformin group with the behavioral weight loss group. Metformin versus control also increased butyrate, acetate, and valerate at 6 months (but not at 12 months). Behavioral weight loss versus control did not significantly alter microbiota composition but did increase acetate at 6 months (but not at 12 months). Increases in acetate were associated with decreases in fasting insulin. Additional whole-genome metagenomic sequencing of a subset of the metformin group showed that metformin altered 62 metagenomic functional pathways, including an acetate-producing pathway and three pathways in glucose metabolism. CONCLUSIONS Metformin, but not behavioral weight loss, impacted gut microbiota composition at 6 months and 12 months. Both metformin and behavioral weight loss altered circulating SCFAs at 6 months, including increasing acetate, which correlated with lower fasting insulin. Future research is needed to elucidate whether the gut microboime mediates or modifies metformin’s health effects.