Saflufenacil (Kixor™) is a new herbicide of the pyrimidinedione chemical class for preplant burndown and selective preemergence dicot weed control in multiple crops, including corn. In this study, the mode of action of saflufenacil was investigated. For initial characterization, a series of biotests was used in a physionomics approach for comprehensive physiological profiling of saflufenacil effects. With the use of treated duckweed plants, metabolite profiling was performed based on quantification of metabolite changes, relative to untreated controls. Physiological and metabolite profiling suggested a mode of action similar to inhibitors of protoporphyrinogen IX oxidase (PPO) in tetrapyrrole biosynthetic pathway. Saflufenacil inhibited PPO enzyme activity in vitro with 50% inhibition of 0.4 nM for the enzymes isolated from black nightshade, velvetleaf, and corn. PPO inhibition by saflufenacil caused accumulations of protoporphyrin IX (Proto) and hydrogen peroxide (H2O2) in leaf tissue of black nightshade and velvetleaf. In corn, only slight increases in Proto and H2O2 were found, which reflects in planta tolerance of this crop. The results show that saflufenacil is a new PPO-inhibiting, peroxidizing herbicide.