Caucasian Old World bluestem (OWB) has escaped into native rangelands and could have unknown effects on distribution, utilization, growth, and reproduction of native plant and animal species. This trial was established to determine the rate and timing of glyphosate application that will provide the greatest OWB suppression. Treatments included glyphosate applied early at the five-leaf stage of growth with 1.12, 2.24, or 3.36 kg glyphosate ai ha−1 (1, 2, or 3 lb ai ac−1), sequential application with an early glyphosate application of 1.12, 2.24, or 3.36 kg ha−1 followed by a second application 8 wk later of either 1.12 or 2.24 kg ha−1, and a late-only application of 1.12, 2.24, or 3.36 kg ha−1. Sequential glyphosate applications with at least one of the early or late applications being 2.24 kg ha−1 or more reduced OWB frequencies from over 87% to below 30% each year. During a moist year, all sequential application treatments reduced OWB frequency to 12% or less. Frequency of OWB the year after application was directly related (r2 = 0.91) to the total amount of glyphosate applied during dry conditions. Seed heads were absent or nearly absent in all sequential application treatments, with the exception of glyphosate at 1.12 kg ha−1 applied early and late during the dry season. Sequential application of glyphosate that includes one treatment either early or late of 2.24 or 3.36 kg ha−1 appears to be the most effective treatment to reduce established OWB during dry years. During years of adequate moisture, a single late application of 2.24 or 3.36 kg ha−1 or sequential applications with 1.12 kg ha−1 at each application is as adequate as sequential applications with greater rates for reducing OWB frequency and achieving OWB control.