Although prenylated isoflavones or glyceollins elicit physiological effects more potent than those by isoflavones, the bioavailability remains unclear. The present study aimed to clarify the intestinal absorption behavior of glyceollins in Sprague–Dawley rats. Upon oral administration of 1.0 mg/kg glyceollin I or III (daidzein as comparative compound) to the rats, no peaks corresponding to the intact forms of the compounds were detected in plasma by liquid chromatography–time-of-flight/mass spectrometry (LC–TOF/MS) analysis. In contrast, enzymatic deconjugation of plasma resulted in successful MS detection of each glyceollin; glyceollin I absorption was >10 times higher than that of daidzein, given its high log P value. The present study demonstrated for the first time that glyceollins were more absorbable than mother isoflavones due to their high hydrophobicity, and they metabolized to form sulfated, glucuronized, and methylated conjugates during the intestinal absorption process.