In the course of screening for herbicidal substances from microbes, we found that a culture filtrate of a soil actinomycete strain exhibited a marked phytotoxic effect against rice seedlings when applied on submerged soil. The active principle was isolated in pure form and identified as isoxazole-4-carboxylic acid, with its structure confirmed by synthesis—this is the first report of isoxazole-4-carboxylic acid as a microbial metabolite and possessing herbicidal activity. The producing strain, isolated from a soil sample collected in Suwa, Nagano Prefecture, was classified as Streptomyces sp. RK-489 based on morphological traits (gray aerial mycelium, brown soluble pigment, Rectiflexibiles spore chains with smooth spores), physiological characteristics (carbohydrate utilization), and chemical properties (L,L-diaminopimelic acid cell wall type I, MK-9 (H6) menaquinone). Fermentation was performed in a jar fermentor using a medium containing glucose, soluble starch, meat extract, dry yeast, soybean meal, and NaCl, with inoculum from a 2-day seed culture. The compound was purified via Amberlite IRA-45, active charcoal, and Sephadex G-10 column chromatography, yielding the sodium salt as a white amorphous powder. The molecular formula of the sodium salt was established as C₄H₂NO₃Na by elementary analysis and HR-EI-MS of the free acid (molecular ion peak 113.0114, corresponding to C₄H₃NO₃). The structure was deduced from UV (λmax 215nm in water), ¹H NMR (singlets at δ 8.45 and 8.84ppm in DMSO-d₆), ¹³C NMR (four signals at δ 122.5, 151.0, 161.2, 168.2ppm in CD₃OD), and IR (carboxylate peaks at 1580 and 1400cm⁻¹) data, and confirmed by synthesis (condensation of ethyl 2,2'-diformylacetate with hydroxylamine followed by acid hydrolysis). The compound demonstrated phytotoxicity against rice seedlings at a rate of 7.5–15g a.i./10a, with herbicidal activity against other plants under investigation. It showed no antimicrobial activity against tested bacteria, fungi, or yeasts, and no toxic signs in mice at an oral dose of 200mg/kg.