Cruciferous vegetables are characterized by high amounts of glucosinolates (GLSs) that are hydrolysed to isothiocyanates (ITCs) and other phytochemicals. The aim of the study was to verify the effect of broccoli intake on plasma levels of carotenoids, vitamins and ITCs and on glutathione S-transferase (GST) activity. Twenty healthy subjects, characterized for GSTM1 and GSTT1 genotype, participated in a cross-over intervention study (broccoli diet versus cruciferous-free diet). Subjects consumed a daily portion of broccoli (10 days, 200 g) providing glucosinolates (200 micromol non-indolyl ITCs evaluated by cyclocondensation reaction after myrosinase treatment), vitamin C (about 100 mg) and carotenoids (about 5 mg lutein and beta-carotene). An increase of folate, carotenoids and ITC plasma concentrations was found. The increase of plasma ITC concentration was independent of the GST genotype. Broccoli intervention did not affect plasma GST activity. Broccoli is a bioavailable source of diverse compounds whose effects on endogenous defence systems deserve further investigation.