4''-Deoxy-4''-episubstituted arbekacin derivatives and 4''-epi-5-deoxy-5-episubstituted arbekacin derivatives were designed and synthesized. Arbekacin and 4''-epiarbekacin both displayed the same antibacterial activity against Staphylococcus aureus (including methicillin-resistant S. aureus (MRSA)) and Pseudomonas aeruginosa. The 4''-epi-5-deoxy-5-episubstituted arbekacin derivatives showed potent antibacterial activity. Among them, the antibacterial activity of 5,4''-diepiarbekacin was superior to that of arbekacin or 5-episubstituted arbekacin against Gram-positive and Gram-negative bacteria. The 6'-N-methyl derivative of the 5,4''-diepiarbekacin was effective against P. aeruginosa expressing an aminoglycoside-modifying enzyme AAC(6')-Ib.