It is well accepted that L-glutamic acid, an excitatory amino acid acting as a neurotransmitter in most parts of the brain, induces neuronal cell death following brain ischemic attack and generates oxygen radicals through various intracellular cascades. In our screening for suppressors of glutamate toxicity to ameliorate brain ischemic injury using neuronal hybridoma N18-RE-105 cells as an in vitro model, we isolated espicufolin (1). This paper reports the fermentation of its producing organism Streptomyces sp. cu39 (cultured in a medium containing glucose 2.5%, soybean meal 1.5%, dried yeast 0.2%, and CaCO3 0.4% at 27°C for 5 days), isolation (mycelial acetone extraction, EtOAc partitioning, silica gel column chromatography, preparative TLC, Sephadex LH-20 chromatography, and HPLC purification to obtain a yellow powder), and structure determination of 1. The molecular formula of 1 was determined as C22H18O6 by high-resolution FAB-MS. Its structure, elucidated via UV/visible spectra, 1H and 13C NMR (DQF-COSY, HMBC, etc.), consists of an anthraquinone moiety and a butyl γ-pyrone moiety linked by an ether bond. In the activity evaluation using N18-RE-105 cells, 1 suppressed L-glutamate toxicity with an EC50 of 40 nM. Since L-glutamate toxicity in these cells is thought to involve glutathione depletion, we tested its effect on buthionine sulfoximine (BSO) toxicity (related to oxygen radicals); however, 1 did not suppress BSO toxicity, suggesting its mode of action is unrelated to antioxidative activity. Further investigation of its biological activity is ongoing.