In the absence of the plant enzyme myrosinase, such as in cooked broccoli, glucoraphanin is considered to be hydrolyzed by bacteria in the lower gut to produce the bioactive isothiocyanate sulforaphane. Simulated digestion using US Pharmacopeia methods caused no loss of glucoraphanin, confirming that glucoraphanin is not destroyed by digestive enzymes during passage through the digestive tract and is able to reach the rat cecum intact. Introduction of glucoraphanin (150 μmol/kg BW) directly into the cecum resulted in appearance of isothiocyanates in the mesenteric plasma by 120 min. In contrast, introduction of sulforaphane (150 μmol/kg BW) directly into the cecum resulted in the appearance of isothiocyanates in the mesenteric plasma within 15 min. Plasma levels remained constant for over an hour. Anaerobic incubation ex vivo of cecal microbiota from male F344 rats with glucoraphanin resulted in very low levels of the hydrolytic metabolite erucin nitrile, showing that hydrolysis of glucosinolates is carried out by cecal microbiota, but metabolism ex vivo by microbiota did not reflect not reflect metabolism in situ. These data are the first to report direct evidence of hydrolysis of glucoraphanin to sulforaphane in the cecum of rats and to show that sulforaphane is able to cross the cecal enterocyte for systemic absorption.