Background: To identify the optimal interval for repeat prostate-specific antigen (PSA) testing to screen for prostate cancer in healthy adults. Patients and methods: A retrospective cohort study was conducted on 7332 healthy males without prostate cancer at baseline from 2005 to 2008. Participants underwent annual health checkups including PSA testing at the Center for Preventive Medicine in Japan. Participants with high PSA (≥4.0 ng/ml) underwent further examination for prostate cancer. A subgroup analysis was conducted age group (<50 years, ≥50 years). Results: Mean age was 50 years. Mean PSA at baseline was 1.2 ng/ml. In over 50-year group, for those with initial PSA of <1.0, 1.0-1.9, 2.0-2.9, and 3.0-3.9 ng/ml at baseline, the 3-year cumulative incidence of prostate cancer was 0%, 0.1%, 0.3%, and 5.7%, respectively. No prostate cancer was identified in those <50 years, regardless of PSA level. Conclusions: If PSA screening is recommended, males >50 years with PSA of 3.0-3.9 ng/ml at baseline should undergo rescreening at 2 years. For men with PSA <3.0 ng/ml, PSA rescreening at intervals of ≥3 years is appropriate. PSA screening may not be indicated in males of <50 years of age.