During the course of screening for new antibiotics effective against rice blast, we isolated nitropeptin (1), a dipeptide antibiotic containing a nitro group, from the culture broth of Streptomyces xanthochromogenus 6257-MQ (isolated from soil in Matsushima-cho, Miyagi prefecture, Japan and taxonomically classified). This work describes its isolation, structural elucidation, and biological properties. Fermentation was performed in a medium with glycerol, peptone, and CaCO₃, followed by isolation/purification using Amberlite IRA-401, activated charcoal, DEAE-Sephadex A-25, Diaion CHP-20, and Sephadex G-10. Physicochemical analyses (elemental analysis, FAB-MS, ¹H/¹³C NMR) and chemical reactions (reduction, DNP derivatization) revealed nitropeptin's structure as N-L-leucyl-β-nitroglutamic acid. It is a water-soluble, acidic compound with spectral features (e.g., UV absorption at 242 nm in alkaline solution due to aci-nitro tautomerism). Biological studies showed a 94% protective effect against Pyricularia oryzae at 200 ppm in greenhouse tests, weak antibacterial activity on nutrient media but activity against Escherichia coli K-12 on synthetic media (reduced by L-glutamine, suggesting it is a glutamine antimetabolite), and an LD₅₀ of 50–100 mg/kg (iv) in mice. Nitropeptin is a unique natural product with an aliphatic nitro group, rare among known antibiotics.