Many beneficial effects of probiotics have been reported; however, few have focussed on the effects of Lactobacillus, a probiotic, on the bioconversion of isoflavonoids. We hypothesized that Lactobacillus rhamnosus will modify the metabolism of isoflavone. In an in vitro incubation, L. rhamnosus JCM 2771 produced daidzein from daidzin along with genistein. However, daidzin and genistein were not detected in the incubation solution of daidzein with L. rhamnosus. In the fecal suspension from a male equol producer with daidzein, equol was detected in the presence of a low or high concentration of L. rhamnosus. In the fecal incubation with daidzin, the equol concentration increased with an increasing concentration of L. rhamnosus JCM 2771. L. rhamnosus affected the equol production in the in vitro incubation of daidzein with fecal flora from a male equol producer. We demonstrated for the first time that L. rhamnosus JCM 2771 could produce genistein from daidzin and affect the equol production of fecal flora from a male equol producer in vitro.