Rotavirus (RV) infections are a major cause of acute gastroenteritis in children and domestic animals, infecting virtually all children within their first 5 y of life. Infants consuming soy-based infant formula (SBIF) are exposed to high levels of isoflavones that exhibit antiviral activity on numerous viruses in vitro and in vivo. Thus, the hypothesis that isoflavones would inhibit RV infection was tested. All isoflavones at SBIF concentrations were tested individually and as a mixture (MIX). Virus infectivity was assessed in MA-104 cells using a focus forming unit assay. Genistin and MIX significantly reduced RV infectivity by 33-62% and 66-74%, respectively, compared with the control and across a wide range of RV concentrations. When tested without genistin, the MIX lost its anti-RV activity, suggesting that genistin is the biologically active isoflavone in our model. In a dose response assay, genistin significantly reduced RV infectivity at a concentration as low as 30 mumol/L. We investigated several possible mechanisms of action. Isoflavones decreased RV infectivity by modulating virion attachment to the host cells and by modulating a postbinding step. Isoflavones did not alter RV triple-layered structure and genistin did not act through inhibition of protein tyrosine kinases and topoisomerase II or by mimicking the effect of estrogens. To our knowledge, this is the first study showing the inhibition of RV infectivity by isoflavones present in SBIF. The modulation of SBIF isoflavone composition and concentration represents novel nutritional approaches to potentially reduce the severity of RV infection in human and production animals.