The first total synthesis of vineomycin B-2 (1) has been accomplished. The aglycon segment, a vineomycinone B-2 derivative, and the glycon segment, an alpha-L-acurosyl-L-rhodinose derivative, were prepared via C-glycosylation using an unprotected sugar and powerful chemoselective O-glycosylation using a 2,3-unsaturated sugar, respectively, as the key steps. Furthermore, effective and simultaneous introduction of the two glycon moieties to the aglycon part by concentration-controlled glycosylation led to the total synthesis of 1.