Melittin is a natural antimicrobial peptide isolated from bee venom, and the non-specific cytotoxicity and hemolytic activity severely limit its clinical application. Glycosylation of proteins is very common in physiological and biochemical processes and can modulate the interaction of proteins with their target. In this study, eight glycosyl groups were used to modify the arginine of melittin at sites 22 and/or 24, and single and double arginine -glycosylated peptides were designed and synthesized. Among the acquired 24 glycopeptides, MLT-1c, MLT-3c, MLT-1f, MLT-3f, MLT-1g, and MLT-3h were found to possess higher helicity, while MLT-3c, MLT-3f and MLT-3h showed dramatically reduced hemolytic activity, especially MLT-3c, whose HC value is 199.3 μM. MLT-1a, MLT-3a and MLT-2c exhibited improved inhibitory activity against , and the MIC was 4 μg mL. MLT-1e and MLT-2g have the strongest tolerance to trypsase, and MLT-3c has the highest therapeutic index. In general, rhamnosyl-modified melittin MLT-3c could be a potent agent for antibacterial and antitumor therapy with high stability and low hemolytic side effects.