[Objective] Elucidation of the biosynthetic mechanism of the deoxyaminosugar moiety in acarbose from Actinoplanes sp. SE50/110. [Methods] On the basis of BlastP analysis, AcbA, AcbB and AcbV were proposed to be associated with the biosynthesis of deoxyaminosugar moiety. Firstly, the in-frame deletion and trans-complementation of acbA, acbB and acbV were performed in SE50/110 to investigate their involvement in acarbose biosynthesis. Then, AcbA, AcbB and AcbV were heterologously expressed in E. coli BL21(DE3)/pGro7 and purified by Ni affinity chromatography. Finally, using D-glucose-1-phosphate as the starting substrate, the biosynthetic process of deoxyaminosugar moiety was elucidated by enzymatic assay. In addition, the properties of these involved enzymes were characterized. [Results] Deletion mutants of acbA, acbB and acbV in SE50/110 were named as ZD03, ZD04 and ZD05, respectively, all of which lost the productivity of acarbose. And then, the production of acarbose was recovered through trans-complementation of acbA, acbB and acbV in ZD03, ZD04 and ZD05, respectively. In vitro enzymatic analysis suggested that AcbA, a D-glucose-1-phosphate thymidylyltransferase, is responsible for the biosynthesis of dTDP-D-glucose from D-glucose-1-phosphate and dTTP. It showed a K_m of (0.185±0.053) mmol/L and a V_max of (2.366±0.217) μmol/(min·mg) with D-glucose-1-phosphate, as well as a K_m of (4.964±1.089) mmol/L and a V_max of (60.310±5.419) μmol/(min·mg) with dTTP. AcbB, a TDP-D-glucose-4,6-dehydratase, catalyzed the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The K_m and V_max of AcbB are (0.353±0.089) mmol/L and (306.401±28.740) μmol/(min·mg), respectively. AcbV is a dTDP-4-keto-6-deoxy-D-glucose-aminotransferase and catalyzes the transamination of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-amino-4,6-dideoxy-D-glucose using glutamic acid as amino donor. The K_m and V_max of AcbV are (1.411±0.293) mmol/L and (3.447±0.279) μmol/(min·mg), respectively. [Conclusion] This study elucidated the biosynthetic pathway of deoxyaminosugar moiety in acarbose, which paved a solide way for a full elucidation of acarbose biosynthesis. Meanwhile, the characterization of the involved enzymes provided important information for the metabolic engineering of SE50/110 to improve acarbose production.