The potential of juglone a plant naphthoquinone as a natural herbicide on new target, 7-keto-8-amino pelargonic acid synthetase (KAPAS) in the early step of biotin biosynthesis pathway, was performed in vitro and in vivo. Juglone effectively inhibited KAPAS activities in vitro and the was . Foliar application of juglone showed very good herbicidal activity to the eight-tested weed species. Among them, Solanum nigrum was completely controlled at a concentration of with main symptoms of desiccation or burndown. Digitaria sanguinalis and Aeschynomene indica were also sensitive to juglone treatment. All eight weed species were controlled by 90~100% at a concentration of . However, soil application of juglone to Digitaria sanguinalis did not show any herbicidal symptoms. Cellular leakage from cucumber leaf squares treated with juglone increased depending on the concentrations increased from 6.25 to after 24 hours incubation with or without light. However, chlorophyll loss in cucumber leaf squares was negligible. Biotin supplements significantly rescued the inhibition of germination rate of Arabidopsis thaliana seeds previously inhibited by the juglone. Our results suggest that the juglone is a possible environmental friendly herbicide candidate with a new target KAPAS inhibiting activity.