Objective: The aim of this study was to investigate the role of inducible nitric oxide synthase (iNOS) in lethal prostate cancer (PCa) by studying the iNOS immunoreactivity in tumor tissue from men diagnosed with localized PCa. Materials and methods: This study is nested within a cohort of men diagnosed with incidental PCa undergoing transurethral resection of the prostate (the Swedish Watchful Waiting Cohort). To investigate molecular determinants of lethal PCa, men who died from PCa (n = 132) were selected as cases; controls (n = 168) comprised men with PCa who survived for at least 10 years without dying from PCa during follow-up. The immunoreactivity of iNOS in prostate tumor epithelial cells and in cells of the surrounding stroma was scored as low/negative, moderate or high. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) for lethal PCa according to iNOS category. Results: There was no association between iNOS immunoreactivity in stroma and lethal disease. However, when comparing high versus low/negative iNOS immunoreactivity in epithelial cells, the OR for lethal PCa was 3.80 (95% CI 1.45–9.97). Conclusion: Patients with localized PCa have variable outcomes, especially those with moderately differentiated tumors. Identifying factors associated with long-term PCa outcomes can elucidate PCa tumor biology and identify new candidate prognostic markers. These findings support the hypothesis that high iNOS in tumor epithelium of the prostate is associated with lethal disease. © 2018 Acta Chirurgica Scandinavica Society