Deficiency of glutathione S-transferase (GST) or NAD(P)H:quinone oxidoreductase 1 (NQO1) in humans is associated with increased risk of urothelial bladder cancer. Broccoli sprouts are a rich source of several isothiocyanates (ITCs), particularly sulforaphane (SF) which has shown promising chemopreventive activities. We report herein that a broccoli sprout ITC extract significantly induced both GST and NQO1 in cultured bladder cells in vitro and in rat bladder tissues in vivo. The inducer activity of the extract was comparable to that of pure SF on the basis of total ITC concentrations. The bladder was one of the most responsive organs to induction of the enzymes by the extract. Induction of the enzymes by the extract was largely mediated by Nrf2, a transcriptional factor that plays a critical role in the induction of many detoxification enzymes. Moreover, induction of GST and NQO1 in the rat bladder in vivo by the extract was associated with high levels of urinary ITC metabolites, but no toxic effects on the bladder mucosa were detected. In conclusion, broccoli sprout ITC extract is a potent inducer of GST and NQO1 in the bladder and is a promising agent for prevention of bladder cancer.