Abstract: Streptomyces sp. YJ-81 with a herbicidal activity was isolated from the rhizosphere soil of Woodsia ilvensis collected from Erlong Mountain, Harbin. To uncover the active component and its herbicidal activity, the active compound from YJ-81 fermentation broth was isolated and purified by silica gel column chromatography, gel-filtration column chromatography, and semi-preparative high-performance liquid chromatography using herbicidal bioassay-guided fractionation. The active compound 1 was identified as herbimycin A by NMR spectroscopy. The seed germination assays on weeds and crops were performed and the result showed that herbimycin A at 100 mg/L could completely inhibit the seed germination of Setaria viridis and Amaranthus retroflexus, and have a certain inhibitory effect on the seed germination of other weeds (Echinochloa crusgalli, Portulaca oleracea, Chenopodium album) and crops (Oryza sativa, Lycopersicon esculentum, Citrullus lanatus, and Zea mays). The pot experiments showed that herbimycin A significantly inhibited the growth of A. retroflexus, P. oleracea, and C. album, and slightly inhibited the growth of S. viridis, E. crusgalli, and L. esculentum, but did not affect the growth of Z. mays, C. lanatus, and O. sativa. Especially, herbimycin A demonstrated crop safety to Z. mays, because no obvious phytotoxic symptom was observed on the leaves of maize, and no significant difference in the fresh weight of the aerial part and chlorophyll content of maize between the control and drug treatment even with 1000 mg/L of herbimycin A. Therefore, herbimycin A could be exploited as a safe, selective, and post-emergence herbicide in the field of maize.