Abstract: Melittin exhibits high antibacterial potency against drug-resistant bacteria. However, the clinical utility of melittin is limited by its serious hemolytic activity. Thus, the need for developing novel melittin analogues with high antimicrobial activity and low hemolytic activity has grown. We designed, synthesized, and evaluated 20 novel melittin analogues with varying hydrophobic, polar or positively charged amino acids. The results showed that 8 compounds had antimicrobial activity (MIC:1-4 μg·mL^-1) against gram-positive pathogens equal to or better than that of melittin, and 16 compounds had low hemolytic activity (HC₅₀ ≥ 11.9 μg·mL^-1). Compounds 13 (MIC:2-4 μg·mL^-1) and 15 (MIC:1-2 μg·mL^-1) showed equal or better antimicrobial activity against both susceptible and resistant strains of Staphylococcus aureus and Enterococcus faecium compared to melittin (MIC:4 μg·mL^-1). Compound 13 (HC₅₀:24.0±4.3 μg·mL^-1) displayed noticeably decreased hemolytic activity compared to melittin (HC₅₀:5.3±0.4 μg·mL^-1). This work established a base for further study on the structure-activity relationships and structure-toxicity relationships of melittin.