Technical Abstract: Japanese knotweed (Polygonum cuspidatum Sieb. & Zucc.) (JKW) is a highly invasive species that has become a serious problem in riparian zones and along road and railroad right-of-ways. Once established, it forms solid colonies that usually choke out all other herbaceous vegetation, displacing native species, negatively affecting wildlife, and altering hydrological processes. We evaluated the ability of six native species mixtures to prevent re-colonization by JKW at a site receiving either one or two years of glyphosate applications and mowing to suppress existing JKW stands. One year of spraying and mowing was not sufficient to adequately suppress JKW. By 37 months after sowing only a multi-species riparian buffer mixture (RBM) had plant cover greater than 20%, whereas, cover for all other mixtures was less than 10%. Japanese knotweed had successfully re-invaded all plots with percent cover ranging from 72 to 96%. Two years of spraying and mowing reduced JKW percent cover to less than 15% for the first two years after sowing and to 28 to 43% by 37 months. Only two species mixtures adequately established when sown following two years of JKW suppression, the RBM and a mixture of Virginia wildrye (Elymus virginicus) and prairie cordgrass (Spartina pectinata). Percent cover for both mixtures was greater than 80% at 25 months after sowing and 50% or more after 37 months. Two years of JKW suppression was necessary before native species mixtures could successfully compete against JKW re-colonization.