Increased circulating serum gamma-glutamyltransferase (GGT) has been linked with an increased risk of chronic disease outcomes, including overall and several site-specific cancers. However, the relationship of GGT with prostate cancer risk is uncertain. We aimed to assess the prospective association of GGT with risk of prostate cancer. Serum GGT activity was assessed at baseline in the Finnish Kuopio Ischemic Heart Disease prospective cohort of 2,390 men aged 42-61 years without a history of cancer at baseline. We corrected for within-person variability in GGT values using data from repeat measurements taken several years apart. During a median follow-up of 24.6 years, 230 cases of prostate cancer occurred. The age-adjusted regression dilution ratio for loge GGT was 0.69 [95% confidence interval (CI): 0.63-0.74]. Serum GGT was nonlinearly associated with risk of prostate cancer. In age-adjusted Cox regression analysis, the hazard ratio (95% CIs) for prostate cancer in a comparison of the top quartile versus bottom quartiles 1-3 of GGT values was 1.43 (1.07 to 1.93; p = 0.017), which persisted on adjustment for several established cancer risk factors 1.46 (1.06 to 2.02; p = 0.020). The association remained unchanged on further adjustment for total energy intake, socioeconomic status, physical activity and C-reactive protein. The association did not importantly vary across several clinical subgroups. GGT is positively and independently associated with future risk of prostate cancer in a middle-aged Finnish male population over long-term follow-up. Further research is needed to understand the mechanistic pathways involved and if GGT may have potential relevance in prostate cancer prevention.