Objective: Transrectal ultrasound guided biopsy for diagnostic workup for prostate cancer (PCa) has a substantial false negative rate. We sought to estimate PCa incidence and mortality following negative biopsy in a cohort of men undergoing prostate cancer screening. Subjects and Methods: The Prostate, Lung, Colorectal and Ovarian (PLCO) cancer screening trial randomized participants 55-74 years to an intervention vs control arm. Intervention arm men received annual prostate-specific antigen (PSA) tests for 6 years and digital rectal exams (DRE) for 4 years. We examined the cohort of men with a positive PSA (> 4 ng/mL) or DRE screen followed within one year by a negative biopsy. PCa incidence and mortality rates from time of first negative biopsy were analyzed as a function of PSA level at diagnosis and other factors. Cumulative incidence and mortality rates accounting for competing risk were estimated. Multivariate proportional hazards regression was utilized to estimate hazard ratios (HRs) of PCa outcomes by PSA level, controlling for age and race. Results: The negative biopsy cohort included 2855 men. Median (25th/75th) age at biopsy was 65 (61/69) years; biopsies occurred between 1994 and 2006. Median (25/75th) follow-up was 13.2 (6.5/16.8) years for incidence and 16.6 (12.3/19.2) years for mortality. 740 PCa cases were diagnosed, with 33 PCa deaths. Overall 20-year cumulative PCa incidence and mortality rates were 26.4% (95% CI: 24.8-28.1) and 1.2% (95% CI: 0.9-1.7), respectively. HRs for PCa incidence and mortality increased significantly with increasing PSA. Conclusion: The mortality rate from PCa through 20 years following a negative biopsy is low.