In the course of screening for substances that modulate immune responses, we found a novel immunosuppressive substance named metacytofilin (MCF) in the culture filtrate of strain TA2759 which was isolated from a soil sample collected in Enzan city, Yamanashi Prefecture, Japan. The strain was identified as Metarhizium sp. TA2759. The structure of MCF was determined by X-ray crystallographic analysis to be 3a-hydroxy-6β-methylamino-6a-(2 methylpropyl)-3β-phenylmethyl-4H-2,3,5,6-tetrahydro-1,4-oxazine-2,5-dione. Although MCF exhibited an immunosuppressive effect on the mixed lymphocyte culture reaction and immune responses in mice, it had low toxicity and no antimicrobial activity. In this communication, the production, isolation, physicochemical properties, structure and biological activity of MCF are reported.