Cathelicidins are effector molecules of vertebrate immunity that play vital roles against microbial invasion. They are widely identified in mammals, but few have been reported in Crocodilians, which are considered to be species with a powerful immune system. In the present study, we identified and characterized a novel cathelicidin from the blood of the Siamese crocodile, Crocodylus siamensis. A cDNA sequence (501 base pair) encoded a predicted 166-residue prepropeptide of C. siamensis cathelicidin (Cs-CATH), which comprised a 21-residue signal peptide, a 109-residue cathelin domain, and a 36-residue mature cathelicidin peptide. Multiple sequence alignment and phylogenetic analysis demonstrated that Cs-CATH shared a high degree of similarity with other crocodilian cathelicidins. Joint consideration of elastase cleavage site, physicochemical properties, and predicted secondary structure demonstrated that RN15 peptide is a candidate antimicrobial peptide derived from Cs-CATH. The synthetic RN15 peptide demonstrates antimicrobial activity against Gram-positive and Gram-negative bacteria. Scanning electron microscopy illustrated RN15-peptide-induced bacteria cells exhibited morphological change. Besides, RN15 peptide demonstrates low hemolytic activity against human erythrocytes and low cytotoxic activity against normal human dermal fibroblasts. This is the first cathelicidin identified from C. siamensis, and it is highlighted that its derived peptide from cathelin domain promises potent novel peptide antibiotics templates.