Background Glucosinolates are a group of phytochemicals that are abundant in cruciferous vegetables and precursors of the potentially chemopreventive isothiocyanates. Isothiocyanates may reduce oxidative stress and inflammation, but little is known regarding the association between glucosinolate intake and risk of type 2 diabetes (T2D). Objective To evaluate the association between the intake of glucosinolates and the incidence of T2D in US men and women. Design This prospective cohort study investigated 200,907 women and men [71,256 women from the Nurses' Health Study (NHS; 1984-2012), 88,293 women from the NHS II (1991-2013), and 41,358 men from the Health Professionals Follow-Up Study (1986-2012)] who were free of diabetes, cardiovascular disease, and cancer at baseline. Diet was assessed using validated semiquantitative food frequency questionnaires. Self-reported T2D incidence was confirmed by a supplementary questionnaire. Results During follow-up in the 3 cohorts, we accumulated 4,303,750 person-years and 16,567 incident cases of T2D. After adjustment for major lifestyle and dietary risk factors for T2D, participants in the highest quintile of total glucosinolate intake had a 19% higher risk (95% CI: 13%, 25%; Ptrend < 0.001) of T2D than did those in the lowest quintile. The intake of 3 major glucosinolate subtypes was consistently and significantly associated with T2D risk, with pooled HRs ranging from 1.13 to 1.18 (all Ptrend < 0.001). A significant association was also observed between total cruciferous vegetable consumption and T2D (HR: 1.16; 95% CI :1.07, 1.25; Ptrend < 0.001). These associations persisted in subgroups defined by demographic, lifestyle, and other dietary factors. Conclusions Dietary glucosinolate intake was associated with a moderately higher risk of T2D in US adults. These results need to be replicated in further investigations, including biomarker-based studies. Mechanistic research is also needed to understand the relation between exposures to glucosinolates, isothiocyanates, and other metabolites with T2D risk. This trial was registered at clinicaltrials.gov as NCT03366532.