Temporins are a kind of small, hydrophobic and C-terminus amidated antimicrobial peptides from Rana species. They are effective against bacteria, fungi, yeast, protozoa and viruses. Two novel temporins named as temporin-La(LLRHVVKILEKYL_amide) and temporin-Lb(LFRHVVKIFEKYL_amide) were cloned from Lithobates catesbeianus. Synthetic peptides of temporin-La and temporin-Lb showed strong antimicrobial activities against bacteria tested, especially Gram-positive bacteria. Besides, temporin-La showed no haemolytic activity to rabbit erythrocytes at the concentration of 250 mg/L while temporin-Lb showed weak haemolytic activity(LC₅₀ ≈ 230 μmol/L). Transmission electron microscopy showed that temporin-La and temporin-Lb induced different effects on bacterial structure of Staphylococcus aureus.