Introduction The Janssen Ad26.COV2.S vaccine was developed to prevent SARS-CoV-2 illness. Phase 3 trials were initiated in 2020, including sites at the University of Kentucky, Norton Healthcare Louisville, and Baptist Health Lexington. This study followed non-immunosuppressed adult subjects through July 2022 to evaluate vaccine efficacy and safety, with a total of 1,094 participants. Methods Subjects aged 18 years or older, in good health, and without major immunological conditions were included. Three trials (ENSEMBLE-1, ENSEMBLE-2, and AMPLIFY) were conducted. Participants were evaluated for vaccine efficacy if they were nucleocapsid antibody-negative and had no prior COVID-19 infection at the time of their first booster. Vaccine regimens included a prime and single boost with Ad26.COV2.S or other combinations of prime and boost vaccinations, including mRNA vaccines. Results A total of 1,094 subjects were enrolled, with 903 subjects evaluated for vaccine efficacy. Among these, 431 received only a prime and single boost with Ad26.COV2.S, and 472 received other combinations of vaccines. During the study period, 174 COVID-19 infections occurred, with 95 (55%) being asymptomatic. The incidence of infections was similar between those who received only Ad26.COV2.S (94 infections) and those with other vaccination regimens (80 infections), with no significant difference (P > .05). There were no serious vaccine-related safety concerns, and only one hospitalization occurred, involving a 75-year-old diabetic who recovered without intubation. Conclusion The Ad26.COV2.S vaccine, as a prime and single boost, demonstrated similar efficacy to other vaccination regimens in preventing COVID-19 infections. Both regimens provided protection against severe disease and hospitalization, with no significant safety concerns identified. This study supports the effectiveness and safety of the Ad26.COV2.S vaccine in the adult population during a period of high COVID-19 incidence.