Cyclin/CDK complexes belong to a serine/threonine protein kinase family and play key roles as the positive regulators in cell cycle progression. Overexpression of cyclins or CDKs, and loss or decreased level of endogenous CDK inhibitor proteins such as p16 and p27 in various tumors have been reported. Thus, the CDKs are considered as new molecular targets for cancer chemotherapy. We established a novel cell-based assay using the budding yeast in which Xenopus cyclin A1 was induced and then CDK (Cdc28) kinase activity was elevated. The hyper-activation of CDK in yeast resulted in showing growth arrest phenotype. The compounds which can rescue the cyclin A1-induced growth arrest might be the new antitumor drug candidates acting on the cyclin/CDK-mediated cell cycle regulation. In the course of our microbial screening program, a novel Streptomyces metabolite belactosin A was identified as an active compound by which regrowth of the growth-arrested yeast was induced. The producing organism KYI 1780 was isolated from a soil sample collected in Kanagawa prefecture, Japan and assigned to the Streptomyces sp. Physico-chemical properties analysis revealed that belactosin A possessed an unique structure containing a novel amino acid, 3-(2-aminocyclopropyl)-alanine (AcpAla), and a β-lactone. Belactosin A did not show antimicrobial activity against either Gram-negative and Gram-positive bacteria at the concentration up to 0.1mg/ml. As for antitumor activity, belactosin A showed in vitro antiproliferative activity against HeLa S3 cells with IC50 value of 51 μM after 72 hours exposure. Low potency of belactosin A on antiproliferative activity was considered to be due to low permeability into human cells, and protection of the carboxylic acid with several esters potentiated antiproliferative activity and antitumor activity in vivo. Further examination on the cell cycle distribution showed a decrease in G1 phase and increase in G2/M phase of cycling HeLa S3 cells in a dose dependent manner after 24 hours exposure. Thus, we carried out microbial screening for their ability to restore the cyclin A1-induced growth arrest in budding yeast and identified a novel natural product belactosin A which inhibited cell cycle progression of human tumor cells at G2/M phase.