Our search for new anthracycline analogs by mutation of rhodomycin-producing Streptomyces violaceus A262 has provided several blocked mutants producing various glycosyl anthracycline compounds, mostly 7-O-glycosyl or 7,10-O-glycosyl derivatives, with few 10-O-glycosyl ones. Since natural 10-O-glycosyl anthracyclines are rare and their antitumor activities have not been intrinsically evaluated, we were interested in 10-O-rhodosaminyl β-rhodomycinone (β-RMN) and β-isorhodomycinone (β-iso-RMN). We found that the 10-O-glycosyl bond is slightly more stable to acid hydrolysis than the 7-O-glycosyl bond when 7,10-O-diglycosyl anthracyclines were subjected to acid hydrolysis, thus attempted direct acid treatment of the culture broth of obelmycin-producing S. violaceus strain SE2-2385 to obtain 10-O-glycosyl anthracyclines. This paper deals with the isolation of 10-O-rhodosaminyl β-RMN (named A262-6) and 10-O-rhodosaminyl β-iso-RMN (named obelmycin H), elucidation of their structures (via physico-chemical properties, HRFAB-MS, UV, IR, NMR, and analysis of aglycone and sugar components), and assessment of their in vitro cytotoxicities against cultured murine leukemic L1210 cells, which showed poorer activity compared with their corresponding 7-O-glycosyl analogs (β-rhodomycin I and obelmycin A).