Abstract: Acarbose, one of the main drugs for type 2 diabetes, currently is produced by Actinoplanes sp. SE50/110 in the industry. Whereas some important intracellular steps of acarbose biosynthesis have been elucidated, less attention has been paid to the extracellular pathways, which would lead to the accumulation of acarbose analogs. In this work, in-frame deletions of acbD, acbE and acbZ were performed in Actinoplanes sp. SE50/110, and their involvements in the biosynthesis of acarbose and its analogs were analyzed by HPLC and α-amylase-starch-iodine（ASI） assay. As detected by HPLC, the titer of acarbose decreased by 42% in the acbD deleted mutant and not significantly changed in the acbE or acbZ deleted mutants. However, as analyzed by the ASI assay, the accumulation of acarbose analogs decreased to 14% with acbE deletion and increased to 375% with acbZ deletion. Furthermore, the combinatory deletions of acbE and acbD resulted in a decrease of acarbose analogs accumulation to 9% compared with the wild-type strain. This might provide us a derivative strain with a clean background for high throughput screening using ASI assay. As a summary, three genes belonging to the extracellular metabolism of acarbose that can cause the accumulation of acarbose and its analogs were preliminarily investigated in this study, which would pave a way for the full elucidation of the extracellular acarbose metabolic pathway.