This study was designed to explore the molecular mechanism of stachyose in enhancing the gastrointestinal stability and absorption of soybean genistein in mice. Male Kunming mice in each group (n = 8) were administered by intragastric gavage with saline, stachyose (250 mg/kg·bw), genistein (100 mg/kg·bw), and stachyose (50, 250, and 500 mg/kg·bw) together with genistein (100 mg/kg·bw) for 4 consecutive weeks, respectively, and then their urine, feces, blood, gut, and liver were collected. UPLC-qTOF/MS analysis showed that levels of genistein and its metabolites (dihydrogenistein, genistein 7-sulfate sodium salt, genistein 4’-β-D-glucuronide, and genistein 7-β-D-glucuronide) in serum and urine were increased with an increase in stachyose dosages in mice. Furthermore, the feces level of genistein aglycone was also elevated by co-treatment of stachyose with genistein. However, the feces concentration of dihydrogenistein, a characteristic metabolite of genistein by gut microorganism, was decreased by stachyose administration in a dose-dependent manner. Additionally, the simultaneous administration with stachyose and genistein in mice could decrease intestinal SULT, UGT, P-gp, and MRP1 expression, relative to the treatment with individual stachyose or genistein. These results demonstrate that stachyose-mediated inhibition against the intestinal degradation of genistein and expression of phase II enzymes and efflux transporters can largely contribute to the elevated bioavailability of soybean genistein.