BACKGROUND: Clostridium difficile causes toxin-mediated nosocomial diarrhea and community-acquired infections; no preventive vaccine is licensed. In this phase 2 study, we explored safety, tolerability, and immunogenicity in older US adults of an investigational bivalent C. difficile vaccine that contains equal dosages of genetically and chemically detoxified toxins A and B. METHODS: Conducted from July 2015 through March 2017, 855 healthy adults aged 65-85 years from 15 US centers were randomized 3:3:1 to receive vaccine (100 or 200 μg) or placebo at 0, 1, and 6 months (month regimen) or 1, 8, and 30 days (day regimen). Serum toxin A- and B-specific neutralizing antibodies were measured. Participant-reported local reactions (LRs) and systemic events (SEs), adverse events (AEs), serious AEs, newly diagnosed chronic medical conditions, and immediate AEs were recorded. RESULTS: The 200-μg dose level elicited higher immune responses than the 100-µg dose level across regimens. Compared with the day regimen, the month regimen induced stronger and more persistent immune responses that remained elevated 12 months after dose 3. Responses peaked at month 7 (month regimen) and day 37 (day regimen). LRs (primarily injection site pain) were more frequent in vaccine recipients than controls; SE frequency was similar across groups. More related AEs were reported in the day regimen group than the month regimen group. CONCLUSIONS: The C. difficile vaccine was safe, well tolerated, and immunogenic in healthy US adults aged 65-85 years. Immune responses were particularly robust in the 200-μg month regimen group. These results support continued vaccine development. CLINICAL TRIALS REGISTRATION: NCT02561195.