Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.