FR225659 and four related compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids. They were isolated from the culture broth of Helicomyces sp. No. 19353 and can be purified by absorptive resin and reverse-phase column chromatography. They are potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents. To identify novel gluconeogenesis inhibitors, we used primary rat hepatocytes to screen various microbial products for their ability to inhibit glucose production in vitro. During the course of this screening, we discovered a novel inhibitor that we denoted FR225659. This compound and four of its related compounds, which also potently inhibit gluconeogenesis in vitro, share a very unique structure that consists of a novel acyl-group and three abnormal amino acids. FR225659 originated from the cultured broth of fungal strain No. 19353, which was isolated from a decayed leaf. In this paper, we describe the taxonomy of fungal strain No. 19353, how it is fermented, and how FR225659 and its four related compounds are isolated. We also characterized the physico-chemical properties of FR225659 and its related compounds.