Variability in glyphosate efficacy has been observed following late day field applications, but the influence of this “time-of-day effect” on weed control and soybean yield is unknown. Additionally, the basis for differences in weed control due to application time of day has not been fully elucidated. In field trials, broadleaf weed biomass was ≥5-fold greater when glyphosate was applied at 6:00 A.M. compared to 6:00 P.M. in three of four site–years. No consistent time-of-day effect was observed on treated grass weeds. Soybean yield was unaffected by treatments, and was similar to the weed-free control. In a greenhouse study, both barnyardgrass and velvetleaf biomass were as much as 25 to 80% greater when glyphosate was applied at 8:00 P.M. vs. 2:00 P.M. Examination of individual components of the time-of-day effect for velvetleaf indicated that leaf angle and time of application accounted for 82 and 18%, respectively, of the biomass change. This research suggests that diurnal changes in leaf movement of velvetleaf account for much of the time-of-day effect, with the remainder likely due to an unknown physiological component.