bstract Crocodilians are regarded as possessing a powerful immune system. However, the composition and action of the crocodilian immune system has remained unclear until now. Cathelicidins, the principal family of host defense peptides (HDPs), play pivotal roles in vertebrate immune defense against microbial invasions. However, cathelicidins from crocodilians have not been extensively studied to date. In the present study, six novel cathelicidins (As-CATH1~6) were identified and characterized from the endangered Chinese Alligator (Alligator sinensis). As-CATH1~6 exhibit no sequence similarity with any of the known cathelicidins. Structure analysis indicated that AsCATH1~3 adopt a random coil secondary conformation, while As-CATH4~6 were predicted to mainly adopt an amphipathic α-helix conformation. Among them, As-CATH4~6 exhibited potent, broadspectrum and rapid antimicrobial activity by inducing the disruption of cell membrane integrity. They also exhibited strong ability to prevent the formation of bacterial biofilms and eradicate preformed biofilms. Furthermore, As-CATH4~6 exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines in mouse peritoneal macrophages. They directly neutralized the LPS toxicity and therefore inhibited the binding of LPS to TLR4 receptor and the subsequent activation of inflammatory response pathways. In a peritonitis mice model, As-CATH2~6 provided effective protection against bacterial infection through enhanced immune cell recruitment. In the host Chinese Alligator, As-CATH1~6 are mainly expressed in immune organs and epithelial tissues. Bacterial infection significantly enhances their expression, which implies an important role in host anti-infective response. Taken together, the diversity and multiple functions of As-CATH1~6 partially reveal the powerful immune system of the Chinese Alligator.