Glucosinolates (GLS) are secondary plant metabolites occurring in cruciferous vegetables. Their biologically active break-down products show cancer preventive properties in animal and cell studies. So far, epidemiologic studies, using consumption of cruciferous vegetables as proxy for GLS intake, yielded inconsistent results. Here, we evaluated the association between dietary intake of GLS in comparison with consumption data of GLS-containing foods and the risk of prostate cancer. The study population comprised 11,405 male participants of the prospective EPIC-Heidelberg cohort study. During a mean follow-up time of 9.4 years, 328 incident cases of prostate cancer occurred. At recruitment, habitual food consumption was assessed by a validated food frequency questionnaire, and intake of individual GLS was estimated by means of a newly compiled database on food content of GLS. Adjusted hazard ratios (HR) for prostate cancer were calculated using the Cox proportional hazard model. Median daily intake of total GLS was 7.9 mg/day (interquartile range 5.1-11.9 mg/day). The risk of prostate cancer decreased significantly over quartiles of total GLS intake (multivariate HR [4th vs. 1st quartile] 0.68, 95% CI 0.48-0.97, p(trend) 0.03). Associations with GLS-containing food intake were weaker. Among GLS subgroups, aliphatic GLS showed the strongest inverse association with cancer risk. Analyses stratified by tumor stage and grade gave hint to inverse associations for localized and low-grade cancers. This study shows an inverse association between dietary intake of GLS and the risk of prostate cancer. Because this is the first prospective study using individual GLS intake data, confirmation in other studies is warranted.